Novel bispecific polypeptides against cd137

ABSTRACT

The invention provides bispecific polypeptides comprising a first binding domain, designated B1, which is capable of binding specifically to CD137, and a second binding domain, designated B2, which is capable of specifically binding to a tumour cell-associated antigen. Also provided are pharmaceutical compositions of such bispecific polypeptides and uses of the same in medicine.

BACKGROUND

Immunotherapy of Cancer

Cancer is a leading cause of premature deaths in the developed world.Immunotherapy of cancer aims to mount an effective immune responseagainst tumour cells. This may be achieved by, for example, breakingtolerance against tumour antigen, augmenting anti-tumor immuneresponses, and stimulating local cytokine responses at the tumor site.The key effector cell of a long lasting anti-tumor immune response isthe activated tumor specific effector T cell. Potent expansion ofactivated tumour-specific effector T cells can redirect the immuneresponse towards the tumor. In this context, various immunosuppressivemechanisms induced by the tumor microenvironment suppress the activityof effector T cells. Several immunosuppressive mediators are expressedby the tumor cells. Such mediators inhibit T cell activation, eitherdirectly, or indirectly by inducing e.g. regulatory T cells (Treg) ormyeloid-derived suppressor cells. Depleting, inhibiting, reverting orinactivating such regulatory cells may therefore provide anti-tumoreffects and revert the immune suppression in the tumor microenvironment.Further, incomplete activation of effector T cells by, for example,dendritic cells can result in sub-optimally activated or anergic Tcells, resulting in an inefficient anti-tumor response. In contrast,adequate induction by dendritic cells can generate a potent expansion ofactivated effector T cells, redirecting the immune response towards thetumor. In addition, Natural killer (NK) cells play an important role intumor immunology by attacking tumor cells with down-regulated humanleukocyte antigen (HLA) expression and by inducing antibody dependentcellular cytotoxicity (ADCC). Stimulation of NK cells may thus alsoreduce tumor growth.

Tumour-Associated Antigens

Tumor-associated antigens (TAA) are cell surface proteins selectivelyexpressed on tumor cells. The term tumor-associated indicates that TAAare not completely tumor-specific, but are rather over-expressed on thetumor. A vast number of TAA have been described and used in varioustherapeutic rationales, including monoclonal antibodies, T cellredirecting therapies with TAA-CD3 bispecific antibodies,immunocytokines and antibody drug conjugates. Some well-studied TAAinclude the EGFR family molecules (HER2, HER3 and EGFR/HER1), VEGFR,EpCAM, CEA, PSA, PSMA, EphA2, gp100, GD2, MUC1, CD20, CD19, CD22 andCD33, summarized in (Cheever et al., 2009).

5T4 (also designated trophoblast glycoprotein, TPBG, M6P1 and Waif1) isa well-defined TAA originally identified by Professor Peter Stern,University of Manchester (Hole and Stern, 1988). It is an oncofetalantigen expressed in a high proportion of patients in a variety ofmalignancies, including non-small cell lung, renal, pancreas, prostate,breast, colorectal, gastric, ovarian and cervix cancers as well as inacute lymphocytic leukemia, and has also been shown to be expressed intumor-initiating cells (Castro et al., 2012; Damelin et al., 2011;Elkord et al., 2009; Southall et al., 1990).

5T4 expression is tumor-selective, with no or low expression in mostnormal tissues. In non-malignant tissue, 5T4 is mainly expressed in theplacenta (trophoblast and amniotic epithelium) and at low levels in somespecialised epithelia (Hole and Stern, 1988), as well as low at levelsin other normal tissues (see US 2010/0021483). However, although lowlevels have been detected in some healthy tissue, the safety riskassociated with this is considered low since expression levels in thetumor are considerably higher. This is supported by the fact that thephase III clinical programs, ANYARA and TroVax targeting 5T4 did notreport severe 5T4-related toxicities.

Data from Stern et al. demonstrate that 5T4 regulates the functionalactivity of CXCR4 (Castro et al., 2012; Southgate et al., 2010). 5T4binding antibodies or 5T4 knock-down resulted in inhibition ofCXCR4-mediated cellular migration. The CXCR4 pathway is involved intumor growth and metastasis. Therefore, targeting 5T4 in a CXCR4inhibitory manner is likely to reduce tumor growth and/or spread.

CD137

CD137 (4-1BB, TNFRSF9) is a tumor necrosis factor (TNF) receptor (TNFR)superfamily member. Its role in cancer immunotherapy has been reviewedin e.g. (Bartkowiak and Curran, 2015). Activation of CD137 is dependenton receptor oligomerization (Rabu et al., 2005; Wyzgol et al., 2009)which is induced by binding to CD137L expressed as a trimer on the cellsurface of antigen presenting cells (APCs) and other cell types. CD137is expressed on various cell populations including activated CD4⁺ andCD8⁺ T cells, regulatory T cells (Treg), dendritic cells (DC),monocytes, mast cells, eosinophils and tumor endothelial cells. CD137activation plays an important role in CD8⁺ T cell activation andsurvival (Lee et al., 2002; Pulle et al., 2006). It sustains andaugments effector functions and preferentially supports Th1 cytokineproduction (Shuford et al., 1997). In CD4⁺ T cells, CD137 stimulationinitially results in activation and later in activation-induced celldeath, which is thought to explain why CD137 agonistic antibodies haveshown therapeutic effect in tumor immunity as well as in autoimmunity(Zhang, J C I, 2007, Sun, Trends Mol Med, 2003). CD137 has also beenreported to suppress Treg function or convert Tregs to cytotoxic CD4⁺T-cells (Akhmetzyanova et al., 2016; So et al., 2008).

CD137 is upregulated on NK cells activated by cytokines or CD16(FcγRIII) stimulation (ref in Melero, CCR 19 (5)1044-53, 2013).Activation of CD137 has been shown to increase antibody-dependentcellular cytotoxicity (ADCC) activity of NK cells in both murine andhuman cells (Kohrt 2012 and 2014 J Clin Invest, reviewed by Hout 2012,Oncoimm). Further, CD137 is expressed on APCs, such as DCs andmacrophages, and stimulation of CD137 on these cell types may induceimmune activation that can result in tumor immunity.

Agonistic CD137 antibody has been shown to activate endothelial cells inthe tumor environment, leading to upregulation of ICAM-1 and VCAM-1 andimproved T cell recruitment (Palazon, Cancer Res, 2011).

Several studies have demonstrated induction of tumor immunity bytreatment with agonistic CD137 mAb in pre-clinical models. The mode ofaction may include various cell types, with CD8⁺ T cells being one ofthe main effector cells involved in CD137-induced tumor immunity (Dubrotet al., 2010; Gauttier et al., 2014; Kim et al., 2001; McMillin et al.,2006; Melero et al., 1997; Miller et al., 2002; Sallin et al., 2014;Taraban et al., 2002; Uno et al., 2006; Vinay and Kwon, 2012; Wilcox etal., 2002). In addition, it synergizes with several immunomodulators,including CpG, TRAIL, CD40, OX-40, DR5, PD-1/PD-L1, CTLA-4, Tim-3, IL-2and IL-12 (Curran et al., 2011; Gray et al., 2008; Guo et al., 2013;Kwong et al., 2013; Lee et al., 2004; Morales-Kastresana et al., 2013;Pan et al., 2002; St Rose et al., 2013; Uno et al., 2006; Wei et al.,2013; Westwood et al., 2010; Westwood et al., 2014a; Westwood et al.,2014b). An important role of CD137 in the induction and maintenance oftumor immunity is further supported by the findings indicating thatCD137+tumor infiltrating T cells are tumor-specific and effectivelyprotect from tumor growth (Ye et al., 2014).

Two CD137 antibodies are in clinical development. Urelumab (BMS-66513)is a fully human IgG4 antibody developed by Bristol-Myers Squibb.Several phase I and II studies in various indications are currentlyongoing. A Phase II study with Urelumab as a second line therapy inmetastatic melanoma was terminated in 2009 due to liver toxicity(Garber, 2011; Li and Liu, 2013). PF-05082566 is a fully human IgG2antibody developed by Pfizer. It is currently in phase I development inlymphoma and various solid cancers and preliminary data suggest that itis well tolerated but with only modest anti-tumor effects.

Toxicity upon CD137 activation has been observed in patients as well asin mouse models (Ascierto et al., 2010; Dubrot et al., 2010; Niu et al.,2007). The toxicity includes skin toxicities and liver toxicitiesmanifested as increased aspartate amino transferase/alanine aminotransferase ratio (ASAT/ALAT) levels and cytokine release. This suggeststhat either the toxicity requires CD137 mediated pre-activation ofimmune cell populations (likely T cells) or it depends on secondaryeffects caused by antidrug-antibodies (ADA) response, potentiallyforming aggregates of CD137 antibodies that may lead to enhancedcross-linking. The toxicities seen in mice are reversible and seem todepend on TNFa/CD8+cells (Ascierto et al., 2010). Toxicology studies inmonkeys showed that both single and repeated dosing of up to 100mg/kgonce weekly for four weeks was tolerable with no skin or liver toxicitydetected (Ascierto 2010 Semin Onc).

TNFR family members are dependent on receptor cross-linking foractivation to be induced. Such crosslinking may either be induced by thenatural ligand expressed on the cell surface of cells or by recombinant,multimerized ligand. Alternatively, it may be induced by an antibodybinding to the receptor and cross-linked by its Fc region bound to anFcγ receptor (FcγR). This cross-linking dependence has been shown forvarious TNFR members, including DR5, GITR, CD27 and CD40 (Li andRavetch, 2011; White et al., 2011; Wilson et al., 2011a; Wilson et al.,2011b; Wyzgol et al., 2009). An important role for the inhibitoryFcγRIIB (CD32B) in activation by agonistic TNFR family antibodies wasshown in some studies (Li and Ravetch, 2011; White et al., 2011; Whiteet al., 2013) whereas other data suggest that activation is induced bycross-linking of inhibitory as well as activating FcγRs (Li and Ravetch,2011; Wilson et al., 2011a).

Similar to other TNFR members, activation of CD137 is dependent onreceptor oligomerization. Hexamers of CD137L effectively induce CD137activation, whereas monomeric or trimeric CD137L does not (Rabu et al.,2005; Wyzgol et al., 2009). Thus, it is likely that CD137 agonisticantibodies require cross-linking, e.g. via FcγR for effective activationto occur in vivo. However, in contrast to other TNFR members, FcγRII isnot critical for induction of tumor immunity by CD137, whereas FcγRIIIimpairs tumor immunity (Sallin et al., 2014; Sanmamed et al., 2015) inmouse studies.

The translational relevance of the role of various FcγR in activation ofCD137 and other TNFR superfamily members is uncertain, since the humanFcγR distribution as well as the affinity of different IgG isotypes todifferent FcγR differ between mice and humans.

Despite progress in the development of immunotherapies for the treatmentof various cancers over the last decade, there remains a need for newand efficacious agents.

Accordingly, the present invention seeks to provide improvedpolypeptide-based therapies for the treatment of cancer.

SUMMARY OF THE INVENTION

A first aspect of the invention provides a bispecific polypeptidecomprising a first binding domain, designated B1, which is capable ofbinding specifically to CD137, and a second binding domain, designatedB2, which is capable of specifically binding to a tumour cell-associatedantigen.

Such bispecific compounds comprising one immune-activating moiety, e.g.a CD137 agonist, and one tumor-targeting moiety, e.g. a 5T4 binder, canbe used to establish a highly effective and safe cancer immunotherapy.

Various types of tumor-localizing immunotherapeutic molecules, such asimmunocytokines and bispecific antibodies have shown beneficial immuneactivation and inhibition of tumor growth in preclinical studies as wellas in the clinic (reviewed in Kiefer and Neri, 2016).

To avoid systemic toxicity by CD137 activating agents, yet obtain highefficacy in the tumor area, the designs of the molecular format of aCD137 agonist may be optimised. For example, a good efficacy/safetyprofile can be obtained by a TAA-CD137 bispecific antibody that requirescrosslinking by binding to the TAA for CD137 activation to occur. Then,pre-activated, CD137-expressing T cells residing in the tumor willpreferentially be activated, whereas CD137 expressing cells in othertissues will not. This would allow focused activation of the relevant,tumor-specific T cells while limiting toxicity induced by generalisedCD137 activation (‘activation’ in this context being a net immuneactivation that results in a tumor-directed T cell response, for exampleby down-regulation of Tregs suppressive function and/or upregulation ofeffector T cell function).

The clinical progress with immunocytokines has so far not beenimpressive and the side effects still remain since the tumor-bindingentity only confers limited tumor localization, with the bulk of theimmunocytokine ending up in other compartments. Bispecific antibodiesthat restrict the activity to the tumor as described in this inventionwould provide a clear advantage over immunocytokines since they areinactive in the absence of tumors.

Further, the bispecific polypeptides of the invention provide a distinctadvantage over bispecific antibodies targeting CD3. CD3-targetingbispecific molecules use T cells as effector cells and are capable ofactivating T cells independent of TAA binding. Thus, they do notactivate tumor specific T-cells in particular. The resulting anti-tumoreffects are therefore not likely to generate a long lasting anti-tumorimmunity. In addition, since CD3 is expressed on all T cells, systemic Tcell activation is associated with toxicity issues. In contrast, thebispecific antibodies of the invention have the potential to selectivelyactivate tumor specific T-cells and generate a long lasting tumourimmunity.

Structure of Bispecific Polypeptide

A “polypeptide” is used herein in its broadest sense to refer to acompound of two or more subunit amino acids, amino acid analogs, orother peptidomimetics. The term “polypeptide” thus includes shortpeptide sequences as well as longer polypeptides and proteins. As usedherein, the term “amino acid” refers to either natural and/or unnaturalor synthetic amino acids, including both D or L optical isomers, andamino acid analogs and peptidomimetics.

The term “bispecific” as used herein means the polypeptide is capable ofspecifically binding at least two target entities.

In one preferred embodiment, the polypeptide is a bispecific antibody(numerous examples of which are described in detail below).

Thus, the first and/or second binding domains may be selected from thegroup consisting of antibodies and antigen-binding fragments thereof.

By “an antibody or an antigen-binding fragment thereof” we includesubstantially intact antibody molecules, as well as chimaericantibodies, humanised antibodies, isolated human antibodies, singlechain antibodies, bispecific antibodies, antibody heavy chains, antibodylight chains, homodimers and heterodimers of antibody heavy and/or lightchains, and antigen-binding fragments and derivatives of the same.Suitable antigen-binding fragments and derivatives include Fv fragments(e.g. single chain Fv and disulphide-bonded Fv), Fab-like fragments(e.g. Fab fragments, Fab′ fragments and F(ab)2 fragments), singlevariable domains (e.g. VH and VL domains) and single domain antibodies(dAbs, including single and dual formats [i.e. dAb-linker-dAb], andnanobodies). The potential advantages of using antibody fragments,rather than whole antibodies, are several-fold. The smaller size of thefragments may lead to improved pharmacological properties, such asbetter penetration of solid tissue. Moreover, antigen-binding fragmentssuch as Fab, Fv, ScFv and dAb antibody fragments can be expressed in andsecreted from E. coli, thus allowing the facile production of largeamounts of the said fragments.

In one embodiment, the antigen-binding fragment is selected from thegroup consisting of: Fv fragments (such as a single chain Fv fragment,or a disulphide-bonded Fv fragment), Fab-like fragments (such as a Fabfragment; a Fab′ fragment or a F(ab)₂ fragment) and single domainantibodies.

For example, the first binding domain (B1) and/or the second bindingdomain (B2) may comprise or consist of a Fab fragment.

Alternatively, or in addition, the first binding domain (B1) and/or thesecond binding domain (B2) may comprise or consist of an Fv fragment(such as an scFv or di-sulphide bridged Fv). Where the binding domain isan scFv, the VH and VL regions therein may be joined by a linkersequence, for example:

[SEQ ID NO: 93] GGGGSGGGGSGGGGS

It will be appreciated by persons skilled in the art that such scFvpolypeptides may be glycosylated, for example N-glycosylated, on one ormore amino acid residues.

The phrase “an antibody or an antigen-binding fragment thereof” is alsointended to encompass antibody mimics (for example, non-antibodyscaffold structures that have a high degree of stability yet allowvariability to be introduced at certain positions). Those skilled in theart of biochemistry will be familiar with many such molecules, asdiscussed in Gebauer & Skerra, 2009, Curr Opin Chem Biol 13(3): 245-255(the disclosures of which are incorporated herein by reference).Exemplary antibody mimics include: affibodies (also called Trinectins;Nygren, 2008, FEBS J, 275, 2668-2676); CTLDs (also called Tetranectins;Innovations Pharmac. Technol. (2006), 27-30); adnectins (also calledmonobodies; Meth. Mol. Biol., 352 (2007), 95-109); anticalins (DrugDiscovery Today (2005), 10, 23-33); DARPins (ankyrins; Nat. Biotechnol.(2004), 22, 575-582); avimers (Nat. Biotechnol. (2005), 23, 1556-1561);microbodies (FEBS J, (2007), 274, 86-95); peptide aptamers (Expert.Opin. Biol. Ther. (2005), 5, 783-797); Kunitz domains (J. Pharmacol.Exp. Ther. (2006) 318, 803-809); affilins (Trends. Biotechnol. (2005),23, 514-522); affimers (Avacta Life Sciences, Wetherby, UK).

Also included within the scope of the invention are chimaeric T-cellreceptors (also known as chimaeric T cell receptors, chimaericimmunoreceptors, and chimaeric antigen receptors or CARs) (see Pule etal., 2003, Cytotherapy 5(3):211-26, the disclosures of which areincorporated herein by reference). These are engineered receptors, whichgraft an arbitrary specificity onto an immune effector cell. Typically,CARs are used to graft the specificity of a monoclonal antibody onto a Tcell; with transfer of their coding sequence facilitated by retroviralvectors. The most common form of such molecules is fusions comprising asingle-chain variable fragment (scFv) derived from a monoclonal antibodyfused to CD3-zeta transmembrane and endodomain. When T cells expressthis fusion molecule, they recognize and kill target cells that expressthe transferred monoclonal antibody specificity.

Persons skilled in the art will further appreciate that the inventionalso encompasses modified versions of antibodies and antigen-bindingfragments thereof, whether existing now or in the future, e.g. modifiedby the covalent attachment of polyethylene glycol or another suitablepolymer (see below).

Methods of generating antibodies and antibody fragments are well knownin the art. For example, antibodies may be generated via any one ofseveral methods which employ induction of in vivo production of antibodymolecules, screening of immunoglobulin libraries (Orlandi et al, 1989.Proc. Natl. Acad. Sci. U.S.A. 86:3833-3837; Winter et al., 1991, Nature349:293-299, the disclosures of which are incorporated herein byreference) or generation of monoclonal antibody molecules by cell linesin culture. These include, but are not limited to, the hybridomatechnique, the human B-cell hybridoma technique, and the Epstein-Barrvirus (EBV)-hybridoma technique (Kohler et al., 1975. Nature256:4950497; Kozbor et al., 1985. J. Immunol. Methods 81:31-42; Cote etal., 1983. Proc. Natl. Acad. Sci. USA 80:2026-2030; Cole et al., 1984.Mol. Cell. Biol. 62:109-120, the disclosures of which are incorporatedherein by reference).

Suitable methods for the production of monoclonal antibodies are alsodisclosed in “Monoclonal Antibodies: A manual of techniques”, H Zola(CRC Press, 1988, the disclosures of which are incorporated herein byreference) and in “Monoclonal Hybridoma Antibodies: Techniques andApplications”, J G R Hurrell (CRC Press, 1982, the disclosures of whichare incorporated herein by reference).

Likewise, antibody fragments can be obtained using methods well known inthe art (see, for example, Harlow & Lane, 1988, “Antibodies: ALaboratory Manual”, Cold Spring Harbor Laboratory, New York, thedisclosures of which are incorporated herein by reference). For example,antibody fragments according to the present invention can be prepared byproteolytic hydrolysis of the antibody or by expression in E. coli ormammalian cells (e.g. Chinese hamster ovary cell culture or otherprotein expression systems) of DNA encoding the fragment. Alternatively,antibody fragments can be obtained by pepsin or papain digestion ofwhole antibodies by conventional methods.

It will be appreciated by persons skilled in the art that for humantherapy or diagnostics, human or humanised antibodies are preferablyused. Humanised forms of non-human (e.g. murine) antibodies aregenetically engineered chimaeric antibodies or antibody fragments havingpreferably minimal-portions derived from non-human antibodies. Humanisedantibodies include antibodies in which complementary determining regionsof a human antibody (recipient antibody) are replaced by residues from acomplementary determining region of a non-human species (donor antibody)such as mouse, rat or rabbit having the desired functionality. In someinstances, Fv framework residues of the human antibody are replaced bycorresponding non-human residues. Humanised antibodies may also compriseresidues which are found neither in the recipient antibody nor in theimported complementarity determining region or framework sequences. Ingeneral, the humanised antibody will comprise substantially all of atleast one, and typically two, variable domains, in which all orsubstantially all of the complementarity determining regions correspondto those of a non-human antibody and all, or substantially all, of theframework regions correspond to those of a relevant human consensussequence. Humanised antibodies optimally also include at least a portionof an antibody constant region, such as an Fc region, typically derivedfrom a human antibody (see, for example, Jones et al., 1986. Nature321:522-525; Riechmann et al., 1988, Nature 332:323-329; Presta, 1992,Curr. Op. Struct. Biol. 2:593-596, the disclosures of which areincorporated herein by reference).

Methods for humanising non-human antibodies are well known in the art.Generally, the humanised antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues, often referred to as imported residues, aretypically taken from an imported variable domain. Humanisation can beessentially performed as described (see, for example, Jones et al.,1986, Nature 321:522-525; Reichmann et al., 1988. Nature 332:323-327;Verhoeyen et al., 1988, Science 239:1534-15361; U.S. Pat. No. 4,816,567,the disclosures of which are incorporated herein by reference) bysubstituting human complementarity determining regions withcorresponding rodent complementarity determining regions. Accordingly,such humanised antibodies are chimaeric antibodies, whereinsubstantially less than an intact human variable domain has beensubstituted by the corresponding sequence from a non-human species. Inpractice, humanised antibodies may be typically human antibodies inwhich some complementarity determining region residues and possibly someframework residues are substituted by residues from analogous sites inrodent antibodies.

Human antibodies can also be identified using various techniques knownin the art, including phage display libraries (see, for example,Hoogenboom & Winter, 1991, J. Mol. Biol. 227:381; Marks et al., 1991, J.Mol. Biol. 222:581; Cole et al., 1985, In: Monoclonal antibodies andCancer Therapy, Alan R. Liss, pp. 77; Boerner et al., 1991. J. Immunol.147:86-95, the disclosures of which are incorporated herein byreference).

It will be appreciated by persons skilled in the art that the bispecificpolypeptides, e.g. antibodies, of the present invention may be of anysuitable structural format.

Thus, in exemplary embodiments of the bispecific antibodies of theinvention:

-   -   (a) binding domain B1 and/or binding domain B2 is an intact IgG        antibody (or, together, form an intact IgG antibody);    -   (b) binding domain B1 and/or binding domain B2 is an Fv fragment        (e.g. an scFv);    -   (c) binding domain B1 and/or binding domain B2 is a Fab        fragment; and/or    -   (d) binding domain B1 and/or binding domain B2 is a single        domain antibody (e.g. domain antibodies and nanobodies).

It will be appreciated by persons skilled in the art that the bispecificantibody may comprise a human Fc region, or a variant of a said region,where the region is an IgG1, IgG2, IgG3 or IgG4 region, preferably anIgG1 or IgG4 region.

Engineering the Fc region of a therapeutic monoclonal antibody or Fcfusion protein allows the generation of molecules that are better suitedto the pharmacology activity required of them (Strohl, 2009, Curr OpinBiotechnol 20(6):685-91, the disclosures of which are incorporatedherein by reference).

(a) Engineered Fc Regions for Increased Half-Life

One approach to improve the efficacy of a therapeutic antibody is toincrease its serum persistence, thereby allowing higher circulatinglevels, less frequent administration and reduced doses.

The half-life of an IgG depends on its pH-dependent binding to theneonatal receptor FcRn. FcRn, which is expressed on the surface ofendothelial cells, binds the IgG in a pH-dependent manner and protectsit from degradation.

Some antibodies that selectively bind the FcRn at pH 6.0, but not pH7.4, exhibit a higher half-life in a variety of animal models.

Several mutations located at the interface between the CH2 and CH3domains, such as T250Q/M428L (Hinton et al., 2004, J Biol Chem.279(8):6213-6, the disclosures of which are incorporated herein byreference) and M252Y/S254T/T256E+H433K1N434F (Vaccaro et al., 2005, Nat.Biotechnol. 23(10):1283-8, the disclosures of which are incorporatedherein by reference), have been shown to increase the binding affinityto FcRn and the half-life of IgG1 in vivo.

(b) Engineered Fc Regions for Altered Effector Function

To ensure lack of CD137 activation in the absence of the tumour antigen,the Fc portion of the bispecific antibody should bind with no or verylow affinity to FcγR, since FcγR-mediated crosslinking of a CD137antibody may induce activation. By “very low affinity” we include thatthe Fc portion exhibits at least 10 times reduced affinity to FcγRI,FcγRII and III compared to wild-type IgG1, as determined by theconcentration where half maximal binding is achieved in flow cytometricanalysis of FcγR expressing cells (Hezareh et al., 2001, J Virol,75(24):12161-8) or by FcγR ELISA (Shields et al., 2001, J Biol Chem.276(9):6591-604).

Another factor to take into account is that engagement of FcγR's mayalso induce antibody-dependent cellular cytotoxicity (ADCC),antibody-dependent cellular phagocytosis (ADCP) and complement-dependentcytotoxicity (CDC) of cells coated with antibodies. Thus, to ensuretumor-dependent CD137 activation as well as to avoid depletion of CD137expressing, tumor-reactive T effector cells, the isotype of a TAA-CD137bispecific antibody should preferably be silent.

The four human IgG isotypes bind the activating Fcγreceptors (FcγRI,FcγRIIa, FcγRIIIa), the inhibitory FcγRIIb receptor, and the firstcomponent of complement (C1q) with different affinities, yielding verydifferent effector functions (Bruhns et al., 2009, Blood.113(16):3716-25, the disclosures of which are incorporated herein byreference). IgG1 molecules have the highest affinity and capacity toinduce effector functions, whereas IgG2, IgG3 and IgG4 are lesseffective (Bruhns, 2012; Hogarth and Pietersz, 2012; Stewart et al.,2014) (Wang 2015 Front 1m; Vidarson 2014 Fron Imm). In addition, certainmutations in the Fc region of IgG1 dramatically reduces FcγR affinityand effector function while retaining neonatal FcR (FcRn) interaction(Ju and Jung, 2014; Leabman et al., 2013; Oganesyan et al., 2008;Sazinsky et al., 2008).

The most widely used IgG1 mutants are N297A alone or in combination withD265A, as well as mutations at positions L234 and L235, including theso-called “LALA” double mutant L234A/L235A. Another position describedto further silence IgG1 by mutation is P329 (see US 2012/0251531).

Thus, choosing a mutated IgG1 format with low effector function butretained binding to FcRn may result in a bispecific antibody with5T4-dependent activation of CD137, and exhibiting a favorableefficacy/safety profile and good PK properties.

Advantageously, the polypeptide is incapable of inducing antibodydependent cell cytotoxicity (ADCC), antibody-dependent cellularphagocytosis (ADCP), and/or complement-dependent cytotoxicity (CDC). By“incapable” we include that the ability of the polypeptide to induceADCC, etc., is at least 10-fold lower than compared to wild-type IgG1 asshown by e.g. monocyte-dependent ADCC or CDC assays described by Hezarehet al. 2001 (supra).

In one embodiment, the Fc region may be a variant of a human IgG1 Fcregion comprising a mutation at one or more of the following positions:

-   -   L234, L235, P239, D265, N297 and/or P329.

Advantageously, alanine may be present at the mutated positions(s).

Optionally, the IgG1 variant may be a variant of a human IgG1 Fc regioncomprising mutations L234A and L235A (i.e. the LALA double mutant; seeSEQ ID NO: 86).

It will be appreciated by persons skilled in the art that the bispecificpolypeptides of the invention may be of several different structuralformats (for example, see Chan & Carter, 2016, Nature Reviews Immunology10, 301-316, the disclosures of which are incorporated herein byreference).

In exemplary embodiments, the bispecific antibody is selected from thegroups consisting of:

-   -   (a) bivalent bispecific antibodies, such as IgG-scFv bispecific        antibodies (for example, wherein B1 is an intact IgG and B2 is        an scFv attached to B1 at the N-terminus of a light chain and/or        at the C-terminus of a light chain and/or at the N-terminus of a        heavy chain and/or at the C-terminus of a heavy chain of the        IgG, or vice versa);    -   (b) monovalent bispecific antibodies, such as a DuoBody® (Genmab        AS, Copenhagen, Denmark) or ‘knob-in-hole’ bispecific antibody        (for example, an scFv-KIH, scFv-KIH^(r), a BiTE-KIH or a        BiTE-KIH^(r) (see Xu et al., 2015, mAbs 7(1):231-242);    -   (c) scFv2-Fc bispecific antibodies (such as ADAPTIR™ bispecific        antibodies from Aptevo Therapeutics Inc, Seattle, US);    -   (d) BiTE/scFv2 bispecific antibodies;    -   (e) DVD-Ig bispecific antibodies or other IgG-FAb, FAb-IgG        bispecific antibodies regardless of bivalency or linkers/        connectors employed;    -   (f) DART-based bispecific antibodies (for example, DART₂-Fc,        DART₂-Fc or DART);    -   (g) DNL-Fab₃ bispecific antibodies; and    -   (h) scFv-HSA-scFv bispecific antibodies.

For example, the bispecific antibody may be an IgG-scFv antibody (seeFIG. 1). The IgG-scFv antibody (and, specifically, the scFv domaintherein) may be in either VH-VL or VL-VH orientation. In one embodiment,the scFv may be stabilised by a S-S bridge between VH and VL.

In an alternative embodiment, the bispecific antibody may be an scFv₂-Fcantibody, for example a dimer wherein each polypeptide comprises, fromthe N-terminus to C-terminus, a first scFv, a hinge domain, an Fc domainand a second scFv (see FIG. 1).

In one embodiment, binding domain B1 and binding domain B2 are fuseddirectly to each other.

In an alternative embodiment, binding domain B1 and binding domain B2are joined via a polypeptide linker. For example, a polypeptide linkermay be a short linker peptide between about 10 to about 25 amino acids.The linker is usually rich in glycine for flexibility, as well as serineor threonine for solubility, and can either connect the N-terminus ofthe VH with the C-terminus of the VL, or vice versa.

Thus, the linker may be selected from the group consisting of the aminoacid sequence SGGGGSGGGGS (SEQ ID NO: 87), SGGGGSGGGGSAP (SEQ ID NO:88), NFSQP (SEQ ID NO: 89), KRTVA (SEQ ID NO: 90), GGGSGGGG (SEQ ID NO:91), GGGGSGGGGS, (SEQ ID NO: 92), GGGGSGGGGSGGGGS (SEQ ID NO: 93),THTCPPCPEPKSSDK (SEQ ID NO: 140), GGGS (SEQ ID NO: 141), EAAKEAAKGGGGS(SEQ ID NO: 142), EAAKEAAK (SEQ ID NO: 143), or (SG)m, where m=1 to 7.

In a further embodiment, binding domain B1 and binding domain B2 areseparated by immunoglobulin constant regions (such as an Fc region) on apolypeptide.

The term “amino acid” as used herein includes the standard twentygenetically-encoded amino acids and their corresponding stereoisomers inthe ‘D’ form (as compared to the natural ‘L’ form), omega-amino acidsother naturally-occurring amino acids, unconventional amino acids (e.g.a,a-disubstituted amino acids, N-alkyl amino acids, etc.) and chemicallyderivatised amino acids (see below).

When an amino acid is being specifically enumerated, such as “alanine”or “Ala” or “A”, the term refers to both L-alanine and D-alanine unlessexplicitly stated otherwise. Other unconventional amino acids may alsobe suitable components for polypeptides of the present invention, aslong as the desired functional property is retained by the polypeptide.For the peptides shown, each encoded amino acid residue, whereappropriate, is represented by a single letter designation,corresponding to the trivial name of the conventional amino acid.

In one embodiment, the antibody polypeptides as defined herein compriseor consist of L-amino acids.

It will be appreciated by persons skilled in the art that the antibodypolypeptides of the invention may comprise or consist of one or moreamino acids which have been modified or derivatised.

Chemical derivatives of one or more amino acids may be achieved byreaction with a functional side group. Such derivatised moleculesinclude, for example, those molecules in which free amino groups havebeen derivatised to form amine hydrochlorides, p-toluene sulphonylgroups, carboxybenzoxy groups, t-butyloxycarbonyl groups, chloroacetylgroups or formyl groups. Free carboxyl groups may be derivatised to formsalts, methyl and ethyl esters or other types of esters and hydrazides.Free hydroxyl groups may be derivatised to form O-acyl or O-alkylderivatives. Also included as chemical derivatives are those peptideswhich contain naturally occurring amino acid derivatives of the twentystandard amino acids. For example: 4-hydroxyproline may be substitutedfor proline; 5-hydroxylysine may be substituted for lysine;3-methylhistidine may be substituted for histidine; homoserine may besubstituted for serine and ornithine for lysine. Derivatives alsoinclude peptides containing one or more additions or deletions as longas the requisite activity is maintained. Other included modificationsare amidation, amino terminal acylation (e.g. acetylation orthioglycolic acid amidation), terminal carboxylamidation (e.g. withammonia or methylamine), and the like terminal modifications.

Alternatively, or in addition, one or more amino acid may beglycosylated, such as N-linked glycosylation (in which glycan moietiesare attached to a nitrogen of asparagine or arginine side chains) and/orO-linked glycosylation (in which glycan moieties are attached to thehydroxyl oxygen of serine, threonine, tyrosine, hydroxylysine orhydroxyproline). Methods for the production of glycosylated antibodiesare well known in the art (for example, see Jefferis, 2009, NatureReviews Drug Discovery 8:226-234, the disclosures of which areincorporated herein by reference).

It will be further appreciated by persons skilled in the art thatpeptidomimetic compounds may also be useful. The term ‘peptidomimetic’refers to a compound that mimics the conformation and desirable featuresof a particular peptide as a therapeutic agent.

For example, the said polypeptide includes not only molecules in whichamino acid residues are joined by peptide (—CO—NH—) linkages but alsomolecules in which the peptide bond is reversed. Such retro-inversopeptidomimetics may be made using methods known in the art, for examplesuch as those described in Meziere et al. (1997) J. lmmunol. 159,3230-3237, which is incorporated herein by reference. This approachinvolves making pseudo-peptides containing changes involving thebackbone, and not the orientation of side chains. Retro-inversepeptides, which contain NH—CO bonds instead of CO—NH peptide bonds, aremuch more resistant to proteolysis. Alternatively, the said polypeptidemay be a peptidomimetic compound wherein one or more of the amino acidresidues are linked by a —y(CH₂NH)— bond in place of the conventionalamide linkage.

In a further alternative, the peptide bond may be dispensed withaltogether provided that an appropriate linker moiety which retains thespacing between the carbon atoms of the amino acid residues is used; itmay be advantageous for the linker moiety to have substantially the samecharge distribution and substantially the same planarity as a peptidebond.

It will also be appreciated that the said polypeptide may convenientlybe blocked at its N- or C-terminus so as to help reduce susceptibilityto exo-proteolytic digestion.

A variety of un-coded or modified amino acids such as D-amino acids andN-methyl amino acids have also been used to modify mammalian peptides.In addition, a presumed bioactive conformation may be stabilised by acovalent modification, such as cyclisation or by incorporation of lactamor other types of bridges, for example see Veber et al., 1978, Proc.Natl. Acad. Sci. USA 75:2636 and Thursell et al., 1983, Biochem.Biophys. Res. Comm. 111:166, which are incorporated herein by reference.

In one embodiment, the bispecific polypeptide of the invention iscapable of inducing tumour immunity. This can be tested in vitro in Tcell activation assays, e.g. by measuring IL-2 and IFNγ production.Activation of effector T cells would indicate that a tumour specific Tcell response can be achieved in vivo. Further, an anti-tumour responsein an in vivo model, such as a mouse model would imply that a successfulimmune response towards the tumour has been achieved.

Thus, the bispecific polypeptide may modulate the activity of a targetimmune system cell, wherein said modulation is an increase or decreasein the activity of said cell. Such cells include T cells, dendriticcells and natural killer cells.

The immune system cell is typically a T cell. Thus, the antibody mayincrease the activity of a CD4+ or CD8+ effector T cell, or may decreasethe activity of a regulatory T cell (Treg). In either case, the neteffect of the antibody will be an increase in the activity of effector Tcells, particularly CD8+ effector T cells. Methods for determining achange in the activity of effector T cells are well known and include,for example, measuring for an increase in the level of T cell cytokineproduction (e.g. IFN-γ or IL-2) or an increase in T cell proliferationin the presence of the antibody relative to the level of T cell cytokineproduction and/or T cell proliferation in the presence of a control.Assays for cell proliferation and/or cytokine production are well known.

For example, the polypeptide may be capable of inducing:

-   -   (a) activation of cytotoxic T cells, i.e. CD8+ T cells;    -   (b) activation of helper T cells, i.e. CD4+ T cells;    -   (c) activation of dendritic cells;    -   (d) activation of natural killer cells; and/or    -   (e) reprograming of Tregs into effector T cells (see        Akhmetzyanova et al., 2016, J Immunol. 196(1):484-92).

The polypeptide or binding domains of the invention can also becharacterised and defined by their binding abilities. Standard assays toevaluate the binding ability of ligands towards targets are well knownin the art, including for example, ELISAs, Western blots, RIAs, and flowcytometry analysis. The binding kinetics (e.g., binding affinity) of thepolypeptide also can be assessed by standard assays known in the art,such as by Surface Plasmon Resonance analysis (SPR).

The terms “binding activity” and “binding affinity” are intended torefer to the tendency of a polypeptide molecule to bind or not to bindto a target. Binding affinity may be quantified by determining thedissociation constant (Kd) for a polypeptide and its target. A lower Kdis indicative of a higher affinity for a target. Similarly, thespecificity of binding of a polypeptide to its target may be defined interms of the comparative dissociation constants (Kd) of the polypeptidefor its target as compared to the dissociation constant with respect tothe polypeptide and another, non-target molecule.

The value of this dissociation constant can be determined directly bywell-known methods, and can be computed even for complex mixtures bymethods such as those, for example, set forth in Caceci et al. (Byte9:340-362, 1984; the disclosures of which are incorporated herein byreference). For example, the Kd may be established using a double-filternitrocellulose filter binding assay such as that disclosed by Wong &Lohman (Proc. Natl. Acad. Sci. USA 90, 5428-5432, 1993). Other standardassays to evaluate the binding ability of ligands such as antibodiestowards targets are known in the art, including for example, ELISAs,Western blots, RIAs, and flow cytometry analysis. The binding kinetics(e.g., binding affinity) of the antibody also can be assessed bystandard assays known in the art, such as by Biacore™ system analysis.

A competitive binding assay can be conducted in which the binding of theantibody to the target is compared to the binding of the target byanother, known ligand of that target, such as another antibody. Theconcentration at which 50% inhibition occurs is known as the Ki. Underideal conditions, the Ki is equivalent to Kd. The Ki value will never beless than the Kd, so measurement of Ki can conveniently be substitutedto provide an upper limit for Kd.

Alternative measures of binding affinity include EC50 or IC50. In thiscontext EC50 indicates the concentration at which a polypeptide achieves50% of its maximum binding to a fixed quantity of target. IC50 indicatesthe concentration at which a polypeptide inhibits 50% of the maximumbinding of a fixed quantity of competitor to a fixed quantity of target.In both cases, a lower level of EC50 or IC50 indicates a higher affinityfor a target. The EC50 and IC50 values of a ligand for its target canboth be determined by well-known methods, for example ELISA. Suitableassays to assess the EC50 and IC50 of polypeptides are set out in theExamples.

A polypeptide of the invention is preferably capable of binding to itstarget with an affinity that is at least two-fold, 10-fold, 50-fold,100-fold or greater than its affinity for binding to another non-targetmolecule.

CD137 Binding Domains

The bispecific polypeptides of the invention comprise a binding domain(B1) which is capable of specifically binding to CD137.

Advantageously, binding domain B1 binds to human CD137 with a K_(D) ofless than 10×10⁻⁹M, for example less than 4×10⁻⁹M or less than1.2×10⁻⁹M.

In exemplary embodiments, binding domain B1 comprises:

-   -   (a) the three CDRs of the heavy chain and/or the three CDRs of        the light chain of antibody 1200/1201 (SEQ ID NOs: 54, 55 and 79        and/or SEQ ID NOs: 46, 65 and 72);    -   (b) the three CDRs of the heavy chain and/or the three CDRs of        the light chain of antibody 1202/1203 (SEQ ID NOs: 54, 55 and 80        and/or SEQ ID NOs: 60, 66 and 73);    -   (c) the three CDRs of the heavy chain and/or the three CDRs of        the light chain of antibody 1204/1205 (SEQ ID NOs: 54, 55 and 81        and/or SEQ ID NOs: 61, 67, 74);    -   (d) the three CDRs of the heavy chain and/or the three CDRs of        the light chain of antibody 1214/1215 (SEQ ID NOs: 54, 55 and 82        and/or SEQ ID NOs: 46, 68 and 75);    -   (e) the three CDRs of the heavy chain and/or the three CDRs of        the light chain of antibody 1618/1619 (SEQ ID NOs: 54, 55 and 83        and/or SEQ ID NOs: 62, 69 and 76);    -   (f) the three CDRs of the heavy chain and/or the three CDRs of        the light chain of antibody 1620/1621 (SEQ ID NOs: 54, 55 and 84        and/or SEQ ID NOs: 63, 70, and 77);    -   (g) the three CDRs of the heavy chain and/or the three CDRs of        the light chain of antibody 1626/1627 (SEQ ID NOs: 54, 55 and 85        and/or SEQ ID NOs: 64, 71 and 78);    -   (h) the three CDRs of the heavy chain and/or the three CDRs of        the light chain of antibody 3012/3013 (SEQ ID NOs: 156, 69 and        76 and/or SEQ ID NOs: 158, 155 and 83);    -   (i) the three CDRs of the heavy chain and/or the three CDRs of        the light chain of antibody 3014/3015 (SEQ ID NOs: 62, 69 and 76        and/or SEQ ID NOs: 159, 160 and 83);    -   (j) the three CDRs of the heavy chain and/or the three CDRs of        the light chain of antibody 3016/3017 (SEQ ID NOs: 62, 69 and 76        and/or SEQ ID NOs: 159, 155 and 83);    -   (k) the three CDRs of the heavy chain and/or the three CDRs of        the light chain of antibody 3018/3019 (SEQ ID NOs: 156, 69 and        76 and/or SEQ ID NOs: 158, 161 and 83);    -   (l) the three CDRs of the heavy chain and/or the three CDRs of        the light chain of antibody 3020/3021 (SEQ ID NOs: 156, 69 and        76 and/or SEQ ID NOs: 162, 163 and 83);    -   (m) the three CDRs of the heavy chain and/or the three CDRs of        the light chain of antibody 3022/3023 (SEQ ID NOs: 156, 69 and        76 and/or SEQ ID NOs: 159, 155 and 83);    -   (n) the three CDRs of the heavy chain and/or the three CDRs of        the light chain of antibody 302⁴/₃025 (SEQ ID NOs: 156, 69 and        76 and/or SEQ ID NOs: 54, 55 and 83);    -   (o) the three CDRs of the heavy chain and/or the three CDRs of        the light chain of antibody 3026/3027 (SEQ ID NOs: 156, 69 and        76 and/or SEQ ID NOs: 162, 165 and 83);    -   (p) the three CDRs of the heavy chain and/or the three CDRs of        the light chain of antibody 3028/3029 (SEQ ID NOs: 157, 69 and        76 and/or SEQ ID NOs: 159, 166 and 83);    -   (q) the three CDRs of the heavy chain and/or the three CDRs of        the light chain of antibody 3030/3031 (SEQ ID NOs: 156, 69 and        76 and/or SEQ ID NOs: 54, 166 and 83);    -   (r) the three CDRs of the heavy chain and/or the three CDRs of        the light chain of antibody 3032/3033 (SEQ ID NOs: 156, 69 and        76 and/or SEQ ID NOs: 54, 55 and 83);    -   (s) the three CDRs of the heavy chain and/or the three CDRs of        the light chain of antibody 303⁴/₃035 (SEQ ID NOs: 62, 69 and 76        and/or SEQ ID NOs: 54, 155 and 83); or    -   (t) the three CDRs of the heavy chain and/or the three CDRs of        the light chain of antibody 3036/3037 (SEQ ID NOs: 156, 69 and        76 and/or SEQ ID NOs: 162, 155 and 83).

wherein the numbering of the antibody (e.g. Antibody X/Y) defines theheavy chain variable region (X) and the light chain variable region (Y),respectively (or, where a single number is indicated, the heavy chainvariable region [X] only is defined). Thus, binding domain B1 maycomprise:

-   -   (a) the heavy chain variable region and/or the light chain        variable region of antibody 1200/1201 (SEQ ID NO: 19 and/or SEQ        ID NO: 17);    -   (b) the heavy chain variable region and/or the light chain        variable region of antibody 1202/1203 (SEQ ID NO: 23 and/or SEQ        ID NO: 21);    -   (c) the heavy chain variable region and/or the light chain        variable region of antibody 1204/1205 (SEQ ID NO: 25 and/or SEQ        ID NO: 27);    -   (d) the heavy chain variable region and/or the light chain        variable region of antibody 1214/1215 (SEQ ID NO: 31 and/or SEQ        ID NO: 29);    -   (e) the heavy chain variable region and/or the light chain        variable region of antibody 1618/1619 (SEQ ID NO: 35 and/or SEQ        ID NO: 33);    -   (f) the heavy chain variable region and/or the light chain        variable region of antibody 1620/1621 (SEQ ID NO: 39 and/or SEQ        ID NO: 37);    -   (g) the heavy chain variable region and/or the light chain        variable region of antibody 1626/1627 (SEQ ID NO: 43 and/or SEQ        ID NO: 41);    -   (h) the heavy chain variable region and/or the light chain        variable region of antibody 3012/3013 (SEQ ID NO: 114 and/or SEQ        ID NO: 115);    -   (i) the heavy chain variable region and/or the light chain        variable region of antibody 3014/3015 (SEQ ID NO: 116 and/or SEQ        ID NO: 117);    -   (j) the heavy chain variable region and/or the light chain        variable region of antibody 3016/3017 (SEQ ID NO: 118 and/or SEQ        ID NO: 119);    -   (k) the heavy chain variable region and/or the light chain        variable region of antibody 3018/3019 (SEQ ID NO: 120 and/or SEQ        ID NO: 121);    -   (l) the heavy chain variable region and/or the light chain        variable region of antibody 3020/3021 (SEQ ID NO: 122 and/or SEQ        ID NO: 123);    -   (m)the heavy chain variable region and/or the light chain        variable region of antibody 3022/3023 (SEQ ID NO: 124 and/or SEQ        ID NO: 125);    -   (n) the heavy chain variable region and/or the light chain        variable region of antibody 3024/3025 (SEQ ID NO: 126 and/or SEQ        ID NO: 127);    -   (o) the heavy chain variable region and/or the light chain        variable region of antibody 3026/3027 (SEQ ID NO: 128 and/or SEQ        ID NO: 129);    -   (p) the heavy chain variable region and/or the light chain        variable region of antibody 3028/3029 (SEQ ID NO: 130 and/or SEQ        ID NO: 131);    -   (q) the heavy chain variable region and/or the light chain        variable region of antibody 3030/3031 (SEQ ID NO: 132 and/or SEQ        ID NO: 133);    -   (r) the heavy chain variable region and/or the light chain        variable region of antibody 3032/3033 (SEQ ID NO: 134 and/or SEQ        ID NO: 135);    -   (s) the heavy chain variable region and/or the light chain        variable region of antibody 3034/3035 (SEQ ID NO: 136 and/or SEQ        ID NO: 137); or    -   (t) the heavy chain variable region and/or the light chain        variable region of antibody 3036/3037 (SEQ ID NO: 138 and/or SEQ        ID NO: 139).

It will be appreciated by persons skilled in the art that the bispecificpolypeptides of the invention may alternatively comprise variants of theabove-defined variable regions.

A variant of any one of the heavy or light chain amino acid sequencesrecited herein may be a substitution, deletion or addition variant ofsaid sequence. A variant may comprise 1, 2, 3, 4, 5, up to 10, up to 20,up to 30 or more amino acid substitutions and/or deletions from the saidsequence. “Deletion” variants may comprise the deletion of individualamino acids, deletion of small groups of amino acids such as 2, 3, 4 or5 amino acids, or deletion of larger amino acid regions, such as thedeletion of specific amino acid domains or other features.“Substitution” variants preferably involve the replacement of one ormore amino acids with the same number of amino acids and makingconservative amino acid substitutions. For example, an amino acid may besubstituted with an alternative amino acid having similar properties,for example, another basic amino acid, another acidic amino acid,another neutral amino acid, another charged amino acid, anotherhydrophilic amino acid, another hydrophobic amino acid, another polaramino acid, another aromatic amino acid or another aliphatic amino acid.Some properties of the 20 main amino acids which can be used to selectsuitable substituents are as follows:

Ala, A aliphatic, hydrophobic, neutral Met, M hydrophobic, neutral Cys,C polar, hydrophobic, neutral Asn, N polar, hydrophilic, neutral Asp, Dpolar, hydrophilic, charged (−) Pro, P hydrophobic, neutral Glu, Epolar, hydrophilic, charged (−) Gln, Q polar, hydrophilic, neutral Phe,F aromatic, hydrophobic, neutral Arg, R polar, hydrophilic, charged (+)Gly, G aliphatic, neutral Ser, S polar, hydrophilic, neutral His, Haromatic, polar, hydrophilic, Thr, T polar, hydrophilic, neutral charged(+) Ile, I aliphatic, hydrophobic, neutral Val, V aliphatic,hydrophobic, neutral Lys, K polar, hydrophilic, charged(+) Trp, Waromatic, hydrophobic, neutral Leu, L aliphatic, hydrophobic, neutralTyr, Y aromatic, polar, hydrophobic

Amino acids herein may be referred to by full name, three letter code orsingle letter code.

Preferred “derivatives” or “variants” include those in which instead ofthe naturally occurring amino acid the amino acid which appears in thesequence is a structural analog thereof. Amino acids used in thesequences may also be derivatised or modified, e.g. labelled, providingthe function of the antibody is not significantly adversely affected.

Derivatives and variants as described above may be prepared duringsynthesis of the antibody or by post-production modification, or whenthe antibody is in recombinant form using the known techniques ofsite-directed mutagenesis, random mutagenesis, or enzymatic cleavageand/or ligation of nucleic acids.

Preferably variants have an amino acid sequence which has more than 60%,or more than 70%, e.g. 75 or 80%, preferably more than 85%, e.g. morethan 90 or 95% amino acid identity to a sequence as shown in thesequences disclosed herein (e.g. the VH or VL region sequences, or CDRsequences therein). This level of amino acid identity may be seen acrossthe full length of the relevant SEQ ID NO sequence or over a part of thesequence, such as across 20, 30, 50, 75, 100, 150, 200 or more aminoacids, depending on the size of the full-length polypeptide.

For example, variants of the above CDR sequences may comprise one, twothree, four, five, six, seven, eight or more amino acid mutationsrelative to the reference sequence (such as a deletion, substitutionand/or insertion of an amino acid).

In connection with amino acid sequences, “sequence identity” refers tosequences which have the stated value when assessed using ClustalW(Thompson et al., 1994, Nucleic Acids Res. 22(22):4673-80; thedisclosures of which are incorporated herein by reference) with thefollowing parameters:

Pairwise alignment parameters—Method: accurate, Matrix: PAM, Gap openpenalty: 10.00, Gap extension penalty: 0.10;

Multiple alignment parameters—Matrix: PAM, Gap open penalty: 10.00, %identity for delay: 30, Penalize end gaps: on, Gap separation distance:0, Negative matrix: no, Gap extension penalty: 0.20, Residue-specificgap penalties: on, Hydrophilic gap penalties: on, Hydrophilic residues:GPSNDQEKR. Sequence identity at a particular residue is intended toinclude identical residues which have simply been derivatised.

Thus, in one embodiment binding domain B1 may comprises one or morevariants of the above-defined light chain variable regions and/or saidheavy chain variable regions having at least 90% sequence identitythereto.

In preferred embodiments, binding domain B1 comprises:

-   -   (a) the heavy chain and/or the light chain of antibody        1200/1201;    -   (b) the heavy chain and/or the light chain of antibody        1202/1203;    -   (c) the heavy chain and/or the light chain of antibody        1204/1205;    -   (d) the heavy chain and/or the light chain of antibody        1214/1215;    -   (e) the heavy chain and/or the light chain of antibody        1618/1619;    -   (f) the heavy chain and/or the light chain of antibody        1620/1621;    -   (g) the heavy chain and/or the light chain of antibody        1626/1627;    -   (h) the heavy chain and/or the light chain of antibody        3012/3013;    -   (i) the heavy chain and/or the light chain of antibody        3014/3015;    -   (j) the heavy chain and/or the light chain of antibody        3016/3017;    -   (k) the heavy chain and/or the light chain of antibody        3018/3019;    -   (l) the heavy chain and/or the light chain of antibody        3020/3021;    -   (m)the heavy chain and/or the light chain of antibody 3022/3023;    -   (n) the heavy chain and/or the light chain of antibody        3024/3025;    -   (o) the heavy chain and/or the light chain of antibody        3026/3027;    -   (p) the heavy chain and/or the light chain of antibody        3028/3029;    -   (q) the heavy chain and/or the light chain of antibody        3030/3031;    -   (r) the heavy chain and/or the light chain of antibody        3032/3033;    -   (s) the heavy chain and/or the light chain of antibody        3034/3035; or    -   (t) the heavy chain and/or the light chain of antibody        3036/3037.

For example, binding domain B1 comprises the light chain variable regionand the heavy chain variable region of antibody 1200/1201 (SEQ ID NO: 19and/or SEQ ID NO: 17), or a variant which has more than 60%, or morethan 70%, e.g. 75 or 80%, preferably more than 85%, e.g. more than 90 or95% amino acid identity to SEQ ID NO: 19 and/or SEQ ID NO: 17).

Alternatively, binding domain B1 comprises the light chain variableregion and the heavy chain variable region of antibody 1618/1619 (SEQ IDNO: 35 and/or SEQ ID NO: 33), or a variant which has more than 60%, ormore than 70%, e.g. 75 or 80%, preferably more than 85%, e.g. more than90 or 95% amino acid identity to SEQ ID NO: 35 and/or SEQ ID NO: 33).

Tumour Cell-Targeting Domains

The bispecific polypeptides of the invention further comprise a bindingdomain (B2) which is capable of specifically binding a tumourcell-associated antigen.

By “tumour cell-associated antigen” we include proteins accessible onthe extracellular surface of tumour cells, such that they are accessibleto the bispecific polypeptides of the invention following administrationinto the body. In one embodiment, the tumour cell-associated antigen istumour specific, i.e. it is found exclusively on tumour cells and not onnormal, healthy cells. However, it will be appreciated by personsskilled in the art that the tumour cell-associated antigen may bepreferentially expressed on tumour cells, i.e. it is expressed on tumourcells at a higher level than on normal, healthy cells (thus, expressionof the antigen on tumour cells may be at least five times more than onnormal, healthy cells, for example expression levels on tumour cells ofat least ten times more, twenty times more, fifty time more or greater).

In one embodiment, binding domain B2 binds to a tumour cell-associatedantigen selected from the group consisting of:

-   -   (a) products of mutated oncogenes and tumour suppressor genes;    -   (b) overexpressed or aberrantly expressed cellular proteins;    -   (c) tumour antigens produced by oncogenic viruses;    -   (d) oncofetal antigens;    -   (e) altered cell surface glycolipids and glycoproteins;    -   (f) cell type-specific differentiation antigens;    -   (g) hypoxia-induced antigens;    -   (h) tumour peptides presented by MHC class I;    -   (i) epithelial tumour antigens;    -   (j) haematological tumour-associated antigens;    -   (k) cancer testis antigens; and    -   (l) melanoma antigens.

Thus, the tumour cell-associated antigen may be selected from the groupconsisting of 5T4, CD20, CD19, MUC-1, carcinoembryonic antigen (CEA),CA-125, 0017-1A, E_(p)CAM, HER2, EGFR, HER3, GD2, Podocalyxin, TROP-2,DLK-1, Ox1R, Nectin-4, FAP, EphA2, EphA3, mesothelin, E-cadherin, CD24and VEGFR.

In one embodiment, the tumour cell-associated antigen is an oncofetalantigen. For example, the tumour cell-associated antigen may be 5T4 (forexample, see UniProt Q13641).

In one embodiment, the tumour cell is a solid tumour cell.

For example, the solid tumour may be selected from the groups consistingof renal cell carcinoma, colorectal cancer, lung cancer, prostatecancer, breast cancer, melanomas, bladder cancer, brain/CNS cancer,cervical cancer, oesophageal cancer, gastric cancer, head/neck cancer,kidney cancer, liver cancer, lymphomas, ovarian cancer, pancreaticcancer and sarcomas.

Advantageously, binding domain B2 binds to the tumour cell-associatedantigen with a K_(D) of less than 10×10⁻⁹M, for example less than4×10⁻⁹M or less than 1.2×10⁻⁹M.

In exemplary embodiments, binding domain B2 comprises:

-   -   (a) the three CDRs of the heavy chain and/or the three CDRs of        the light chain of antibody 1206/1207 (SEQ ID NOs: 54, 55 and 56        and/or SEQ ID NOs: 45, 47 and 50);    -   (b) the three CDRs of the heavy chain and/or the three CDRs of        the light chain of antibody 1208/1135 (SEQ ID NOs: 54, 55 and 57        and/or SEQ ID NOs: 46, 48 and 51);    -   (c) the three CDRs of the heavy chain and/or the three CDRs of        the light chain of antibody 1210/1211 (SEQ ID NOs: 54, 55 and 58        and/or SEQ ID NOs: 46, 48 and 52);    -   (d) the three CDRs of the heavy chain and/or the three CDRs of        the light chain of antibody 1212/1213 (SEQ ID NOs: 54, 55 and 59        and/or SEQ ID NOs: 46, 49 and 53);    -   (e) the three CDRs of the heavy chain and/or the three CDRs of        the light chain of antibody 2992/2993 (SEQ ID NOs: 144, 48 and        52 and/or SEQ ID NOs: 145, 55 and 58);    -   (f) the three CDRs of the heavy chain and/or the three CDRs of        the light chain of antibody 2994/2995 (SEQ ID NOs: 146, 147 and        52 and/or SEQ ID NOs: 145, 55 and 58);    -   (g) the three CDRs of the heavy chain and/or the three CDRs of        the light chain of antibody 2996/2997 (SEQ ID NOs: 146, 48 and        52 and/or SEQ ID NOs: 148, 55 and 58);    -   (h) the three CDRs of the heavy chain and/or the three CDRs of        the light chain of antibody 2998/2999 (SEQ ID NOs: 146, 48 and        52 and/or SEQ ID NOs: 149, 55 and 58);    -   (i) the three CDRs of the heavy chain and/or the three CDRs of        the light chain of antibody 3000/3001 (SEQ ID NOs: 150, 48 and        52 and/or SEQ ID NOs: 148, 151 and 58);    -   (j) the three CDRs of the heavy chain and/or the three CDRs of        the light chain of antibody 3002/3003 (SEQ ID NOs: 152, 48 and        52 and/or SEQ ID NOs: 145, 55 and 58);    -   (k) the three CDRs of the heavy chain and/or the three CDRs of        the light chain of antibody 3004/3005 (SEQ ID NOs: 146, 48 and        52 and/or SEQ ID NOs: 153, 55 and 58);    -   (l) the three CDRs of the heavy chain and/or the three CDRs of        the light chain of antibody 3006/3007 (SEQ ID NOs: 144, 48 and        52 and/or SEQ ID NOs: 154, 155 and 58); or    -   (m)the three CDRs of the heavy chain and/or the three CDRs of        the light chain of antibody 3008/3009 (SEQ ID NOs: 146, 48 and        52 and/or SEQ ID NOs: 154, 55 and 58).

wherein the numbering of the antibody (e.g. Antibody X/Y) defines theheavy chain variable region (X) and the light chain variable region (Y),respectively (or, where a single number is indicated, the heavy chainvariable region [X] only is defined).

Thus, binding domain B2 may comprise:

-   -   (a) the heavy chain variable region and/or the light chain        variable region of antibody 1206/1207 (SEQ ID NO: 3 and/or SEQ        ID NO: 1);    -   (b) the heavy chain variable region and/or the light chain        variable region of antibody 1208/1135 (SEQ ID NO: 7 and/or SEQ        ID NO: 5);    -   (c) the heavy chain variable region and/or the light chain        variable region of antibody 1210/1211 (SEQ ID NO: 11 and/or SEQ        ID NO: 9);    -   (d) the heavy chain variable region and/or the light chain        variable region of antibody 1212/1213 (SEQ ID NO: 15 and/or SEQ        ID NO: 13);    -   (e) the heavy chain variable region and/or the light chain        variable region of antibody 2992/2993 (SEQ ID NO: 96 and/or SEQ        ID NO: 97);    -   (f) the heavy chain variable region and/or the light chain        variable region of antibody 2994/2995 (SEQ ID NO: 98 and/or SEQ        ID NO: 99);    -   (g) the heavy chain variable region and/or the light chain        variable region of antibody 2996/2997 (SEQ ID NO: 100 and/or SEQ        ID NO: 101);    -   (h) the heavy chain variable region and/or the light chain        variable region of antibody 2998/2999 (SEQ ID NO: 102 and/or SEQ        ID NO: 103);    -   (i) the heavy chain variable region and/or the light chain        variable region of antibody 3000/3001 (SEQ ID NO: 104 and/or SEQ        ID NO: 105);    -   (j) the heavy chain variable region and/or the light chain        variable region of antibody 3002/3003 (SEQ ID NO: 106 and/or SEQ        ID NO: 107);    -   (k) the heavy chain variable region and/or the light chain        variable region of antibody 3004/3005 (SEQ ID NO: 108 and/or SEQ        ID NO: 109);    -   (l) the heavy chain variable region and/or the light chain        variable region of antibody 3006/3007 (SEQ ID NO: 110 and/or SEQ        ID NO: 111); or    -   (m)the heavy chain variable region and/or the light chain        variable region of antibody 3008/3009 (SEQ ID NO: 112 and/or SEQ        ID NO: 113).

It will be appreciated by skilled persons that binding domain B2 mayalternatively comprise variants of said light chain variable regionsand/or said heavy chain variable regions, for example having at least90% sequence identity thereto.

For example, variants of the above CDR sequences may comprise one, twothree, four, five, six, seven, eight or more amino acid mutationsrelative to the reference sequence (such as a deletion, substitutionand/or insertion of an amino acid).

In one embodiment, binding domain B2 comprises:

-   -   (a) the heavy chain and/or the light chain of antibody        1206/1207;    -   (b) the heavy chain and/or the light chain of antibody        1208/1135;    -   (c) the heavy chain and/or the light chain of antibody        1210/1211;    -   (d) the heavy chain and/or the light chain of antibody        1212/1213;    -   (e) the heavy chain and/or the light chain of antibody        2992/2993;    -   (f) the heavy chain and/or the light chain of antibody        2994/2995;    -   (g) the heavy chain and/or the light chain of antibody        2996/2993;    -   (h) the heavy chain and/or the light chain of antibody        2998/2999;    -   (i) the heavy chain and/or the light chain of antibody        3000/3001;    -   (j) the heavy chain and/or the light chain of antibody        3002/3003;    -   (k) the heavy chain and/or the light chain of antibody        3004/3005;    -   (l) the heavy chain and/or the light chain of antibody        3006/3007; or    -   (m)the heavy chain and/or the light chain of antibody 3008/3009.

For example, binding domain B2 comprises the heavy chain variable regionand the light chain variable region of antibody 1208/1135 (SEQ ID NO: 7and SEQ ID NO: 5), or a variant which has more than 60%, or more than70%, e.g. 75 or 80%, preferably more than 85%, e.g. more than 90 or 95%amino acid identity to SEQ ID NO: 7 and/or SEQ ID NO: 5).

Alternatively, binding domain B2 comprises the heavy chain variableregion and the light chain variable region of antibody 1210/1211 (SEQ IDNO: 11 and SEQ ID NO: 9), or a variant which has more than 60%, or morethan 70%, e.g. 75 or 80%, preferably more than 85%, e.g. more than 90 or95% amino acid identity to SEQ ID NO: 11 and/or SEQ ID NO: 9).

Alternatively, binding domain B2 comprises the heavy chain variableregion and the light chain variable region of antibody 2992/2993 (SEQ IDNO: 96 and SEQ ID NO: 97), or a variant which has more than 60%, or morethan 70%, e.g. 75 or 80%, preferably more than 85%, e.g. more than 90 or95% amino acid identity to SEQ ID NO: 96 and/or SEQ ID NO: 97.

Alternatively, binding domain B2 comprises the heavy chain variableregion and the light chain variable region of antibody 2994/2995 (SEQ IDNO: 98 and SEQ ID NO: 99), or a variant which has more than 60%, or morethan 70%, e.g. 75 or 80%, preferably more than 85%, e.g. more than 90 or95% amino acid identity to SEQ ID NO: 98 and/or SEQ ID NO: 99.

Exemplary CD137-5T4 Bispecific Antibodies

In one preferred embodiment of the bispecific polypeptides of theinvention, binding domain B1 is an IgG and binding domain B2 is an scFv.Conversely, binding domain B1 may be an scFv and binding domain B2 maybe an IgG.

In an alternative embodiment of the bispecific polypeptides of theinvention, binding domain B1 is an scFv and binding domain B2 is an scFv(e.g. in an scFv₂-Fc format).

In exemplary bispecific polypeptides of the invention:

-   -   (a) B1 comprises the three CDRs of the light chain and/or the        three CDRs of the heavy chain of antibody 1200/1201 (SEQ ID NOs:        54, 55 and 79 and/or SEQ ID NOs: 46, 65 and 72) and B2 comprises        the three CDRs of the light chain and/or the three CDRs of the        heavy chain of antibody 1208/1135 (SEQ ID NOs: 54, 55 and 57        and/or SEQ ID NOs: 46, 48 and 51);    -   (b) B1 comprises the three CDRs of the light chain and/or the        three CDRs of the heavy chain of antibody 1200/1201 (SEQ ID NOs:        54, 55 and 79 and/or SEQ ID NOs: 46, 65 and 72) and B2 comprises        the three CDRs of the light chain and/or the three CDRs of the        heavy chain of antibody 1210/1211 (SEQ ID NOs: 54, 55 and 58        and/or SEQ ID NOs: 46, 48 and 52);    -   (c) B1 comprises the three CDRs of the light chain and/or the        three CDRs of the heavy chain of antibody 1618/1619 (SEQ ID NOs:        54, 55 and 83 and/or SEQ ID NOs: 62, 69 and 76) and B2 comprises        the three CDRs of the light chain and/or the three CDRs of the        heavy chain of antibody 1208/1135 (SEQ ID NOs: 54, 55 and 57        and/or SEQ ID NOs: 46, 48 and 51); or    -   (d) B1 comprises the three CDRs of the light chain and/or the        three CDRs of the heavy chain of antibody 1618/1619 (SEQ ID NOs:        54, 55 and 83 and/or SEQ ID NOs: 62, 69 and 76) and B2 comprises        the three CDRs of the light chain and/or the three CDRs of the        heavy chain of antibody 1210/1211 (SEQ ID NOs: 54, 55 and 58        and/or SEQ ID NOs: 46, 48 and 52).

Thus, in certain embodiments:

-   -   (a) B1 comprises the light chain variable region and/or the        heavy chain variable region of antibody 1200/1201 (SEQ ID NO: 19        and/or SEQ ID NO: 17) and B2 comprises the light chain variable        region and/or the heavy chain variable region of antibody        1208/1135 (SEQ ID NO: 7 and/or SEQ ID NO: 5);    -   (b) B1 comprises the light chain variable region and/or the        heavy chain variable region of antibody 1200/1201 (SEQ ID NO: 19        and/or SEQ ID NO: 17) and B2 comprises the light chain variable        region and/or the heavy chain variable region of antibody        1210/1211 (SEQ ID NO: 11 and/or SEQ ID NO: 9);    -   (c) B1 comprises the light chain variable region and/or the        heavy chain variable region of antibody 1618/1619 (SEQ ID NO: 35        and/or SEQ ID NO: 33) and B2 comprises the light chain variable        region and/or the heavy chain variable region of antibody        1208/1135 (SEQ ID NO: 7 and/or SEQ ID NO: 5);    -   (d) B1 comprises the light chain variable region and/or the        heavy chain variable region of antibody 1618/1619 (SEQ ID NO: 35        and/or SEQ ID NO: 33) and B2 comprises the light chain variable        region and/or the heavy chain variable region of antibody        1210/1211 (SEQ ID NO: 11 and/or SEQ ID NO: 9); or    -   (e) variants of said light chain variable regions and/or said        heavy chain variable regions, for example having at least 90%        sequence identity thereto (as discussed above).

In a preferred embodiment, B1 comprises the light chain variable regionand/or the heavy chain variable region of antibody 1200/1201 (SEQ ID NO:19 and/or SEQ ID NO: 17) and B2 comprises the light chain variableregion and/or the heavy chain variable region of antibody 1210/1211 (SEQID NO: 11 and/or SEQ ID NO: 9), or variants of said light chain variableregions and/or said heavy chain variable regions (for example, having atleast 90% sequence identity thereto).

In an alternative preferred embodiment, B1 comprises the light chainvariable region and/or the heavy chain variable region of antibody1210/1211 (SEQ ID NO: 11 and/or SEQ ID NO: 9) and B2 comprises the lightchain variable region and/or the heavy chain variable region of antibody1618/1619 (SEQ ID NO: 35 and/or SEQ ID NO: 33), or variants of saidlight chain variable regions and/or said heavy chain variable regions(for example, having at least 90% sequence identity thereto).

Typically, the bispecific antibody polypeptides of the invention willcomprise constant region sequences, in addition to the above-definedvariable region sequences.

An exemplary heavy chain constant region amino acid sequence which maybe combined with any VH region sequence disclosed herein (to form acomplete heavy chain) is the following IgG1 heavy chain constant regionsequence:

[SEQ ID NO: 94] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPE LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

or a variant thereof comprising the L234A and L235A (“LALA”) mutations(see amino acid residues highlighted above).

Likewise, an exemplary light chain constant region amino acid sequencewhich may be combined with any VL region sequence disclosed herein (toform a complete light chain) is the kappa chain constant region sequencereproduced here:

[SEQ ID NO: 95] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC.

Thus, the bispecific antibody of the invention may comprise:

-   -   (a) a binding domain (B1) comprising a heavy chain variable        region of any of SEQ ID NOs: 17, 21, 27, 29, 33, 37, 41, 114,        116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136 or 138 and        a light chain variable region of any of SEQ ID NOs: 19, 23, 25,        31, 35, 39, 43, 115, 117, 119, 121, 123, 125, 127, 129, 131,        133, 135 or 139;    -   (b) a heavy chain constant region comprising an Fc region (for        example, SEQ ID NO: 94 or 96);    -   (c) a binding domain (B2) comprising a heavy chain variable        region of any of SEQ ID NOs: 1, 5, 9, 13, 96, 98, 100, 102, 104,        106, 108, 110 or 112 and a light chain variable region of any of        SEQ ID NOs: 3, 7, 11, 15, 97, 99, 101, 103, 105, 107, 109, 111        or 113; and    -   (d) optionally, a light chain constant region (for example SEQ        ID NO:95).

In one preferred embodiment, the bispecific antibody of the invention isan IgG-scFv bispecific antibody (for example, wherein B1 is an intactIgG and B2 is an scFv attached to the C-terminus of a heavy chain of theIgG, or vice versa).

For example, the bispecific antibody may comprise the followingcomponents:

-   -   (a) two heavy chains each comprising, in order from the        N-terminus to the C terminus:        -   [a VH sequence]-[an H chain constant region]-[a            connector]-[an scFv]            -   wherein the scFv may comprise of consist of in order                from the N-terminus to the C terminus:            -   [a VH sequence]-[a linker]-[a VL sequence], or vice                versa    -   (b) two light chains each comprising, in order from the        N-terminus to the C terminus:        -   [a VL sequence]-[an L chain constant region]

In such “Morrison format” bispecific antibodies:

-   -   the VH sequences may be selected from any of those disclosed        herein, for example from clone 1618 (SEQ ID NO: 33), clone 1210        (SEQ ID NO:9) or a variant thereof;    -   the H chain constant region may be selected from any of those        disclosed herein, for example SEQ ID NO:86 or 94;    -   the connector may be selected from any of those disclosed        herein, for example SEQ ID NOs:92 or 140 or 143;    -   the linker within the scFv may be selected from any of those        disclosed herein, for example SEQ ID NO:93, and    -   the VL sequence within the scFv may be selected from any of        those disclosed herein, for example from clone 1619 (SEQ ID NO:        35), clone 1211 (11) or a variant thereof; and    -   the L chain constant region may be selected from any of those        disclosed herein, for example SEQ ID NO:95.

As discussed above, methods for the production of antibody polypeptidesof the invention are well known in the art.

Conveniently, the antibody polypeptide is or comprises a recombinantpolypeptide. Suitable methods for the production of such recombinantpolypeptides are well known in the art, such as expression inprokaryotic or eukaryotic hosts cells (for example, see Green &Sambrook, 2012, Molecular Cloning, A Laboratory Manual, Fourth Edition,Cold Spring Harbor, New York, the relevant disclosures in which documentare hereby incorporated by reference).

Antibody polypeptides of the invention can also be produced using acommercially available in vitro translation system, such as rabbitreticulocyte lysate or wheatgerm lysate (available from Promega).Preferably, the translation system is rabbit reticulocyte lysate.Conveniently, the translation system may be coupled to a transcriptionsystem, such as the TNT transcription-translation system (Promega). Thissystem has the advantage of producing suitable mRNA transcript from anencoding DNA polynucleotide in the same reaction as the translation.

It will be appreciated by persons skilled in the art that antibodypolypeptides of the invention may alternatively be synthesisedartificially, for example using well known liquid-phase or solid phasesynthesis techniques (such as t-Boc or Fmoc solid-phase peptidesynthesis).

Polynucleotides, Vectors and Cells

A second aspect of the invention provides an isolated nucleic acidmolecule encoding a bispecific polypeptide according to any one of thepreceding claims, or a component polypeptide chain thereof. For example,the nucleic acid molecule may comprise any of the nucleotide sequencesprovided in Table A.

Thus, a polynucleotide of the invention may encode any polypeptide asdescribed herein, or all or part of B1 or all or part of B2. The terms“nucleic acid molecule” and “polynucleotide” are used interchangeablyherein and refer to a polymeric form of nucleotides of any length,either deoxyribonucleotides or ribonucleotides, or analogs thereof.Non-limiting examples of polynucleotides include a gene, a genefragment, messenger RNA (mRNA), cDNA, recombinant polynucleotides,plasmids, vectors, isolated DNA of any sequence, isolated RNA of anysequence, nucleic acid probes, and primers. A polynucleotide of theinvention may be provided in isolated or substantially isolated form. Bysubstantially isolated, it is meant that there may be substantial, butnot total, isolation of the polypeptide from any surrounding medium. Thepolynucleotides may be mixed with carriers or diluents which will notinterfere with their intended use and still be regarded as substantiallyisolated.

A nucleic acid sequence which “encodes” a selected polypeptide is anucleic acid molecule which is transcribed (in the case of DNA) andtranslated (in the case of mRNA) into a polypeptide in vivo when placedunder the control of appropriate regulatory sequences. The boundaries ofthe coding sequence are determined by a start codon at the 5′ (amino)terminus and a translation stop codon at the 3′ (carboxy) terminus. Forthe purposes of the invention, such nucleic acid sequences can include,but are not limited to, cDNA from viral, prokaryotic or eukaryotic mRNA,genomic sequences from viral or prokaryotic DNA or RNA, and evensynthetic DNA sequences. A transcription termination sequence may belocated 3′ to the coding sequence.

Representative polynucleotides which encode examples of a heavy chain orlight chain amino acid sequence of an antibody may comprise or consistof any one of the nucleotide sequences disclosed herein, for example thesequences set out in Table A.

A suitable polynucleotide sequence may alternatively be a variant of oneof these specific polynucleotide sequences. For example, a variant maybe a substitution, deletion or addition variant of any of the abovenucleic acid sequences. A variant polynucleotide may comprise 1, 2, 3,4, 5, up to 10, up to 20, up to 30, up to 40, up to 50, up to 75 or morenucleic acid substitutions and/or deletions from the sequences given inthe sequence listing.

Suitable variants may be at least 70% homologous to a polynucleotide ofany one of nucleic acid sequences disclosed herein, preferably at least80 or 90% and more preferably at least 95%, 97% or 99% homologousthereto. Preferably homology and identity at these levels is present atleast with respect to the coding regions of the polynucleotides. Methodsof measuring homology are well known in the art and it will beunderstood by those of skill in the art that in the present context,homology is calculated on the basis of nucleic acid identity. Suchhomology may exist over a region of at least 15, preferably at least 30,for instance at least 40, 60, 100, 200 or more contiguous nucleotides.Such homology may exist over the entire length of the unmodifiedpolynucleotide sequence.

Methods of measuring polynucleotide homology or identity are known inthe art. For example the UWGCG Package provides the BESTFIT programwhich can be used to calculate homology (e.g. used on its defaultsettings) (Devereux et al, 1984, Nucleic Acids Research 12:387-395; thedisclosures of which are incorporated herein by reference).

The PILEUP and BLAST algorithms can also be used to calculate homologyor line up sequences (typically on their default settings), for exampleas described in Altschul, 1993, J Mol Evo! 36:290-300; Altschul et al,1990, J Mol Blot 215:403-10, the disclosures of which are incorporatedherein by reference).

Software for performing BLAST analysis is publicly available through theNational Centre for Biotechnology Information(http://www.ncbi.nlm.nih.gov/). This algorithm involves firstidentifying high scoring sequence pair (HSPs) by identifying short wordsof length W in the query sequence that either match or satisfy somepositive-valued threshold score T when aligned with a word of the samelength in a database sequence. T is referred to as the neighbourhoodword score threshold (Altschul et al, supra). These initialneighbourhood word hits act as seeds for initiating searches to findHSPs containing them. The word hits are extended in both directionsalong each sequence for as far as the cumulative alignment score can beincreased. Extensions for the word hits in each direction are haltedwhen: the cumulative alignment score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, Tand X determine the sensitivity and speed of the alignment. The BLASTprogram uses as defaults a word length (W) of 11, the BLOSUM62 scoringmatrix (see Henikoff & Henikoff, 1992, Proc. Natl. Acad. Sci. USA89:10915-10919; the disclosures of which are incorporated herein byreference) alignments (B) of 50, expectation (E) of 10, M=5, N=4, and acomparison of both strands.

The BLAST algorithm performs a statistical analysis of the similaritybetween two sequences; see e.g. Karlin & Altschul, 1993, Proc. Natl.Acad. Sci. USA 90:5873-5787; the disclosures of which are incorporatedherein by reference. One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance. For example, a sequence isconsidered similar to another sequence if the smallest sum probabilityin comparison of the first sequence to the second sequence is less thanabout 1, preferably less than about 0.1, more preferably less than about0.01, and most preferably less than about 0.001.

The homologue may differ from a sequence in the relevant polynucleotideby less than 3, 5, 10, 15, 20 or more mutations (each of which may be asubstitution, deletion or insertion). These mutations may be measuredover a region of at least 30, for instance at least 40, 60 or 100 ormore contiguous nucleotides of the homologue.

In one embodiment, a variant sequence may vary from the specificsequences given in the sequence listing by virtue of the redundancy inthe genetic code. The DNA code has 4 primary nucleic acid residues (A,T, C and G) and uses these to “spell” three letter codons whichrepresent the amino acids the proteins encoded in an organism's genes.The linear sequence of codons along the DNA molecule is translated intothe linear sequence of amino acids in the protein(s) encoded by thosegenes. The code is highly degenerate, with 61 codons coding for the 20natural amino acids and 3 codons representing “stop” signals. Thus, mostamino acids are coded for by more than one codon—in fact several arecoded for by four or more different codons. A variant polynucleotide ofthe invention may therefore encode the same polypeptide sequence asanother polynucleotide of the invention, but may have a differentnucleic acid sequence due to the use of different codons to encode thesame amino acids.

A polypeptide of the invention may thus be produced from or delivered inthe form of a polynucleotide which encodes, and is capable ofexpressing, it.

Polynucleotides of the invention can be synthesised according to methodswell known in the art, as described by way of example in Green &Sambrook (2012, Molecular Cloning—a laboratory manual, 4^(th) edition;Cold Spring Harbor Press; the disclosures of which are incorporatedherein by reference).

The nucleic acid molecules of the present invention may be provided inthe form of an expression cassette which includes control sequencesoperably linked to the inserted sequence, thus allowing for expressionof the polypeptide of the invention in vivo. These expression cassettes,in turn, are typically provided within vectors (e.g., plasmids orrecombinant viral vectors). Such an expression cassette may beadministered directly to a host subject. Alternatively, a vectorcomprising a polynucleotide of the invention may be administered to ahost subject. Preferably the polynucleotide is prepared and/oradministered using a genetic vector. A suitable vector may be any vectorwhich is capable of carrying a sufficient amount of genetic information,and allowing expression of a polypeptide of the invention.

The present invention thus includes expression vectors that comprisesuch polynucleotide sequences. Such expression vectors are routinelyconstructed in the art of molecular biology and may for example involvethe use of plasmid DNA and appropriate initiators, promoters, enhancersand other elements, such as for example polyadenylation signals whichmay be necessary, and which are positioned in the correct orientation,in order to allow for expression of a peptide of the invention. Othersuitable vectors would be apparent to persons skilled in the art (seeGreen & Sambrook, supra).

The invention also includes cells that have been modified to express apolypeptide of the invention. Such cells include transient, orpreferably stable higher eukaryotic cell lines, such as mammalian cellsor insect cells, lower eukaryotic cells, such as yeast or prokaryoticcells such as bacterial cells. Particular examples of cells which may bemodified by insertion of vectors or expression cassettes encoding for apolypeptide of the invention include mammalian HEK293T, CHO, HeLa, NSOand COS cells. Preferably the cell line selected will be one which isnot only stable, but also allows for mature glycosylation and cellsurface expression of a polypeptide.

Such cell lines of the invention may be cultured using routine methodsto produce a polypeptide of the invention, or may be usedtherapeutically or prophylactically to deliver antibodies of theinvention to a subject. Alternatively, polynucleotides, expressioncassettes or vectors of the invention may be administered to a cell froma subject ex vivo and the cell then returned to the body of the subject.

In one embodiment, the nucleic acid molecule encodes an antibody heavychain or variable region thereof.

In one embodiment, the nucleic acid molecule encodes an antibody lightchain or variable region thereof.

By “nucleic acid molecule” we include DNA (e.g. genomic DNA orcomplementary DNA) and mRNA molecules, which may be single- ordouble-stranded. By “isolated” we mean that the nucleic acid molecule isnot located or otherwise provided within a cell.

In one embodiment, the nucleic acid molecule is a cDNA molecule.

It will be appreciated by persons skilled in the art that the nucleicacid molecule may be codon-optimised for expression of the antibodypolypeptide in a particular host cell, e.g. for expression in humancells (for example, see Angov, 2011, Biotechnol. J. 6(6):650-659, thedisclosures of which are incorporated herein by reference).

Also included within the scope of the invention are the following:

-   -   (a) a third aspect of the invention provides a vector (such as        an expression vector) comprising a nucleic acid molecule        according to the second aspect of the invention;    -   (b) a fourth aspect of the invention provides a host cell (such        as a mammalian cell, e.g. human cell, or Chinese hamster ovary        cell, e.g. CHOK1SV cells) comprising a nucleic acid molecule        according to the second aspect of the invention or a vector        according to the third aspect of the invention; and    -   (c) a fifth aspect of the invention provides a method of making        an antibody polypeptide according to the first aspect of the        invention comprising culturing a population of host cells        according to the fourth aspect of the invention under conditions        in which said polypeptide is expressed, and isolating the        polypeptide therefrom.

In a sixth aspect, the present invention provides compositionscomprising molecules of the invention, such as the antibodies,bispecific polypeptides, polynucleotides, vectors and cells describedherein. For example, the invention provides a composition comprising oneor more molecules of the invention, such as one or more antibodiesand/or bispecific polypeptides of the invention, and at least onepharmaceutically acceptable carrier.

It will be appreciated by persons skilled in the art that additionalcompounds may also be included in the pharmaceutical compositions,including, chelating agents such as EDTA, citrate, EGTA or glutathione.

The pharmaceutical compositions may be prepared in a manner known in theart that is sufficiently storage stable and suitable for administrationto humans and animals. For example, the pharmaceutical compositions maybe lyophilised, e.g. through freeze drying, spray drying, spray cooling,or through use of particle formation from supercritical particleformation.

By “pharmaceutically acceptable” we mean a non-toxic material that doesnot decrease the effectiveness of the CD137 and 5T4-binding activity ofthe antibody polypeptide of the invention. Such pharmaceuticallyacceptable buffers, carriers or excipients are well-known in the art(see Remington's Pharmaceutical Sciences, 18th edition, A. R Gennaro,Ed., Mack Publishing Company (1990) and handbook of PharmaceuticalExcipients, 3rd edition, A. Kibbe, Ed., Pharmaceutical Press (2000), thedisclosures of which are incorporated herein by reference).

The term “buffer” is intended to mean an aqueous solution containing anacid-base mixture with the purpose of stabilising pH. Examples ofbuffers are Trizma, Bicine, Tricine, MOPS, MOPSO, MOBS, Tris, Hepes,HEPBS, MES, phosphate, carbonate, acetate, citrate, glycolate, lactate,borate, ACES, ADA, tartrate, AMP, AMPD, AMPSO, BES, CABS, cacodylate,CHES, DIPSO, EPPS, ethanolamine, glycine, HEPPSO, imidazole,imidazolelactic acid, PIPES, SSC, SSPE, POPSO, TAPS, TABS, TAPSO andTES.

The term “diluent” is intended to mean an aqueous or non-aqueoussolution with the purpose of diluting the antibody polypeptide in thepharmaceutical preparation. The diluent may be one or more of saline,water, polyethylene glycol, propylene glycol, ethanol or oils (such assafflower oil, corn oil, peanut oil, cottonseed oil or sesame oil).

The term “adjuvant” is intended to mean any compound added to theformulation to increase the biological effect of the antibodypolypeptide of the invention. The adjuvant may be one or more of zinc,copper or silver salts with different anions, for example, but notlimited to fluoride, chloride, bromide, iodide, tiocyanate, sulfite,hydroxide, phosphate, carbonate, lactate, glycolate, citrate, borate,tartrate, and acetates of different acyl composition. The adjuvant mayalso be cationic polymers such as cationic cellulose ethers, cationiccellulose esters, deacetylated hyaluronic acid, chitosan, cationicdendrimers, cationic synthetic polymers such as poly(vinyl imidazole),and cationic polypeptides such as polyhistidine, polylysine,polyarginine, and peptides containing these amino acids.

The excipient may be one or more of carbohydrates, polymers, lipids andminerals. Examples of carbohydrates include lactose, glucose, sucrose,mannitol, and cyclodextrines, which are added to the composition, e.g.for facilitating lyophilisation. Examples of polymers are starch,cellulose ethers, cellulose carboxymethylcellulose, hydroxypropylmethylcellulose, hydroxyethyl cellulose, ethylhydroxyethyl cellulose,alginates, carageenans, hyaluronic acid and derivatives thereof,polyacrylic acid, polysulphonate, polyethylenglycol/polyethylene oxide,polyethyleneoxide/polypropylene oxide copolymers,polyvinylalcohol/polyvinylacetate of different degree of hydrolysis, andpolyvinylpyrrolidone, all of different molecular weight, which are addedto the composition, e.g., for viscosity control, for achievingbioadhesion, or for protecting the lipid from chemical and proteolyticdegradation. Examples of lipids are fatty acids, phospholipids, mono-,di-, and triglycerides, ceramides, sphingolipids and glycolipids, all ofdifferent acyl chain length and saturation, egg lecithin, soy lecithin,hydrogenated egg and soy lecithin, which are added to the compositionfor reasons similar to those for polymers. Examples of minerals aretalc, magnesium oxide, zinc oxide and titanium oxide, which are added tothe composition to obtain benefits such as reduction of liquidaccumulation or advantageous pigment properties.

The antibody polypeptides of the invention may be formulated into anytype of pharmaceutical composition known in the art to be suitable forthe delivery thereof.

In one embodiment, the pharmaceutical compositions of the invention maybe in the form of a liposome, in which the antibody polypeptide iscombined, in addition to other pharmaceutically acceptable carriers,with amphipathic agents such as lipids, which exist in aggregated formsas micelles, insoluble monolayers and liquid crystals. Suitable lipidsfor liposomal formulation include, without limitation, monoglycerides,diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bileacids, and the like. Suitable lipids also include the lipids abovemodified by poly(ethylene glycol) in the polar headgroup for prolongingbloodstream circulation time. Preparation of such liposomal formulationsis can be found in for example U.S. Pat. No. 4,235,871, the disclosuresof which are incorporated herein by reference.

The pharmaceutical compositions of the invention may also be in the formof biodegradable microspheres. Aliphatic polyesters, such as poly(lacticacid) (PLA), poly(glycolic acid) (PGA), copolymers of PLA and PGA (PLGA)or poly(caprolactone) (PCL), and polyanhydrides have been widely used asbiodegradable polymers in the production of microspheres. Preparationsof such microspheres can be found in U.S. Pat. No. 5,851,451 and in EP 0213 303, the disclosures of which are incorporated herein by reference.

In a further embodiment, the pharmaceutical compositions of theinvention are provided in the form of polymer gels, where polymers suchas starch, cellulose ethers, cellulose carboxymethylcellulose,hydroxypropyl methyl cellulose, hydroxyethyl cellulose,ethylhydroxyethyl cellulose, alginates, carageenans, hyaluronic acid andderivatives thereof, polyacrylic acid, polyvinyl imidazole,polysulphonate, polyethylenglycol/polyethylene oxide,polyethyleneoxide/polypropylene oxide copolymers,polyvinylalcohol/polyvinylacetate of different degree of hydrolysis, andpolyvinylpyrrolidone are used for thickening of the solution containingthe agent. The polymers may also comprise gelatin or collagen.

Alternatively, the antibody polypeptide may simply be dissolved insaline, water, polyethylene glycol, propylene glycol, ethanol or oils(such as safflower oil, corn oil, peanut oil, cottonseed oil or sesameoil), tragacanth gum, and/or various buffers.

It will be appreciated that the pharmaceutical compositions of theinvention may include ions and a defined pH for potentiation of actionof the active antibody polypeptide. Additionally, the compositions maybe subjected to conventional pharmaceutical operations such assterilisation and/or may contain conventional adjuvants such aspreservatives, stabilisers, wetting agents, emulsifiers, buffers,fillers, etc.

The pharmaceutical compositions according to the invention may beadministered via any suitable route known to those skilled in the art.Thus, possible routes of administration include parenteral (intravenous,subcutaneous, and intramuscular), topical, ocular, nasal, pulmonar,buccal, oral, parenteral, vaginal and rectal. Also administration fromimplants is possible.

In one preferred embodiment, the pharmaceutical compositions areadministered parenterally, for example, intravenously,intracerebroventricularly, intraarticularly, intra-arterially,intraperitoneally, intrathecally, intraventricularly, intrasternally,intracranially, intramuscularly or subcutaneously, or they may beadministered by infusion techniques. They are conveniently used in theform of a sterile aqueous solution which may contain other substances,for example, enough salts or glucose to make the solution isotonic withblood. The aqueous solutions should be suitably buffered (preferably toa pH of from 3 to 9), if necessary. The preparation of suitableparenteral formulations under sterile conditions is readily accomplishedby standard pharmaceutical techniques well known to those skilled in theart.

Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents. The formulations may be presented in unit-dose or multi-dosecontainers, for example sealed ampoules and vials, and may be stored ina freeze-dried (lyophilised) condition requiring only the addition ofthe sterile liquid carrier, for example water for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions may be prepared from sterile powders, granules and tabletsof the kind previously described.

Thus, the pharmaceutical compositions of the invention are particularlysuitable for parenteral, e.g. intravenous, administration.

Alternatively, the pharmaceutical compositions may be administeredintranasally or by inhalation (for example, in the form of an aerosolspray presentation from a pressurised container, pump, spray ornebuliser with the use of a suitable propellant, such asdichlorodifluoromethane, trichlorofluoro-methane,dichlorotetrafluoro-ethane, a hydrofluoroalkane such as1,1,1,2-tetrafluoroethane (HFA 134A3 or 1,1,1,2,3,3,3-heptafluoropropane(HFA 227EA3), carbon dioxide or other suitable gas). In the case of apressurised aerosol, the dosage unit may be determined by providing avalve to deliver a metered amount. The pressurised container, pump,spray or nebuliser may contain a solution or suspension of the activepolypeptide, e.g. using a mixture of ethanol and the propellant as thesolvent, which may additionally contain a lubricant, e.g. sorbitantrioleate. Capsules and cartridges (made, for example, from gelatin) foruse in an inhaler or insufflator may be formulated to contain a powdermix of a compound of the invention and a suitable powder base such aslactose or starch.

The pharmaceutical compositions will be administered to a patient in apharmaceutically effective dose. A ‘therapeutically effective amount’,or ‘effective amount’, or ‘therapeutically effective’, as used herein,refers to that amount which provides a therapeutic effect for a givencondition and administration regimen. This is a predetermined quantityof active material calculated to produce a desired therapeutic effect inassociation with the required additive and diluent, i.e. a carrier oradministration vehicle. Further, it is intended to mean an amountsufficient to reduce and most preferably prevent, a clinicallysignificant deficit in the activity, function and response of the host.Alternatively, a therapeutically effective amount is sufficient to causean improvement in a clinically significant condition in a host. As isappreciated by those skilled in the art, the amount of a compound mayvary depending on its specific activity. Suitable dosage amounts maycontain a predetermined quantity of active composition calculated toproduce the desired therapeutic effect in association with the requireddiluent. In the methods and use for manufacture of compositions of theinvention, a therapeutically effective amount of the active component isprovided. A therapeutically effective amount can be determined by theordinary skilled medical or veterinary worker based on patientcharacteristics, such as age, weight, sex, condition, complications,other diseases, etc., as is well known in the art. The administration ofthe pharmaceutically effective dose can be carried out both by singleadministration in the form of an individual dose unit or else severalsmaller dose units and also by multiple administrations of subdivideddoses at specific intervals. Alternatively, the does may be provided asa continuous infusion over a prolonged period.

Particularly preferred compositions are formulated for systemicadministration.

The composition may preferably be formulated for sustained release overa period of time. Thus the composition may be provided in or as part ofa matrix facilitating sustained release. Preferred sustained releasematrices may comprise a montanide or γ-polyglutamic acid (PGA)nanoparticles.

The antibody polypeptides can be formulated at various concentrations,depending on the efficacy/toxicity of the polypeptide being used. Forexample, the formulation may comprise the active antibody polypeptide ata concentration of between 0.1 μM and 1 mM, more preferably between 1 μMand 500 μM, between 500 μM and 1 mM, between 300 μM and 700 μM, between1 μM and 100 μM, between 100 μM and 200 μM, between 200 μM and 300 μM,between 300 μM and 400 μM, between 400 μM and 500 μM, between 500 μM and600 μM, between 600 μM and 700 μM, between 800 μM and 900 μM or between900 μM and 1 mM. Typically, the formulation comprises the activeantibody polypeptide at a concentration of between 300 μM and 700 μM.

Typically, the therapeutic dose of the antibody polypeptide (with orwithout a therapeutic moiety) in a human patient will be in the range of100 μg to 700 mg per administration (based on a body weight of 70 kg).For example, the maximum therapeutic dose may be in the range of 0.1 to10 mg/kg per administration, e.g. between 0.1 and 5 mg/kg or between 1and 5 mg/kg or between 0.1 and 2 mg/kg. It will be appreciated that sucha dose may be administered at different intervals, as determined by theoncologist/physician; for example, a dose may be administered daily,twice-weekly, weekly, bi-weekly or monthly.

It will be appreciated by persons skilled in the art that thepharmaceutical compositions of the invention may be administered aloneor in combination with other therapeutic agents used in the treatment ofcancers, such as antimetabolites, alkylating agents, anthracyclines andother cytotoxic antibiotics, vinca alkyloids, etoposide, platinumcompounds, taxanes, topoisomerase I inhibitors, other cytostatic drugs,antiproliferative immunosuppressants, corticosteroids, sex hormones andhormone antagonists, and other therapeutic antibodies (such asantibodies against a tumour-associated antigen or an immune checkpointmodulator).

For example, the pharmaceutical compositions of the invention may beadministered in combination with an immunotherapeutic agent that binds atarget selected from the group consisting of PD-1/PD-1L, CTLA-4, 0X40,CD40, GITR, LAGS, TIM3, CD27 and KIR.

Thus, the invention encompasses combination therapies comprising abispecific polypeptide of the invention together with a furtherimmunotherapeutic agent, effective in the treatment of cancer, whichspecifically binds to an immune checkpoint molecule. It will beappreciated that the therapeutic benefit of the furtherimmunotherapeutic agent may be mediated by attenuating the function ofan inhibitory immune checkpoint molecule and/or by activating thefunction of a stimulatory immune checkpoint or co-stimulatory molecule.

In one embodiment, the further immunotherapeutic agent is selected fromthe group consisting of:

-   -   (a) an immunotherapeutic agent that inhibits the function of        PD-1 and/or PD-1L;    -   (b) an immunotherapeutic agent that inhibits the function of        CTLA-4;    -   (c) an immunotherapeutic agent that activates the function of        OX40; and    -   (d) an immunotherapeutic agent that binds activates the function        of CD40.

Thus, the further immunotherapeutic agent may be a PD1 inhibitor, suchas an anti-PD1 antibody, or antigen-binding fragment thereof capable ofinhibiting PD1 function (for example, Nivolumab, Pembrolizumab,Lambrolizumab, PDR-001, MEDI-0680 and AMP-224). Alternatively, the PD1inhibitor may comprise or consist of an anti-PD-L1 antibody, orantigen-binding fragment thereof capable of inhibiting PD1 function (forexample, Durvalumab, Atezolizumab, Avelumab and MDX-1105).

In another embodiment, the further immunotherapeutic agent is a CTLA-4inhibitor, such as an anti-CTLA-4 antibody or antigen-binding portionthereof.

In a further embodiment, the further immunotherapeutic agent activatesOX40, such as an agonistic anti-OX40 antibody or antigen-binding portionthereof.

In a further embodiment, the further immunotherapeutic agent activatesCD40, such as an agonistic anti-CD40 antibody or antigen-binding portionthereof.

It will be appreciated by persons skilled in the art that the presenceof the two active agents (as detailed above) may provide a synergisticbenefit in the treatment of a tumour in a subject. By “synergistic” weinclude that the therapeutic effect of the two agents in combination(e.g. as determined by reference to the rate of growth or the size ofthe tumour) is greater than the additive therapeutic effect of the twoagents administered on their own. Such synergism can be identified bytesting the active agents, alone and in combination, in a relevant cellline model of the solid tumour.

Also within the scope of the present invention are kits comprisingpolypeptides or other compositions of the invention and instructions foruse. The kit may further contain one or more additional reagents, suchas an additional therapeutic or prophylactic agent as discussed above.

Medical Uses and Methods

The polypeptides in accordance with the present invention may be used intherapy or prophylaxis. In therapeutic applications, polypeptides orcompositions are administered to a subject already suffering from adisorder or condition, in an amount sufficient to cure, alleviate orpartially arrest the condition or one or more of its symptoms. Suchtherapeutic treatment may result in a decrease in severity of diseasesymptoms, or an increase in frequency or duration of symptom-freeperiods. An amount adequate to accomplish this is defined as“therapeutically effective amount”. In prophylactic applications,polypeptides or compositions are administered to a subject not yetexhibiting symptoms of a disorder or condition, in an amount sufficientto prevent or delay the development of symptoms. Such an amount isdefined as a “prophylactically effective amount”. The subject may havebeen identified as being at risk of developing the disease or conditionby any suitable means.

Thus, a seventh aspect of the invention provides a bispecificpolypeptide according to the first aspect of the invention for use inmedicine.

An eighth aspect of the invention provides a bispecific polypeptideaccording to the first aspect of the invention for use in treating aneoplastic disorder in a subject.

By ‘treatment’ we include both therapeutic and prophylactic treatment ofthe patient. The term ‘prophylactic’ is used to encompass the use of anagent, or formulation thereof, as described herein which either preventsor reduces the likelihood of a neoplastic disorder, or the spread,dissemination, or metastasis of cancer cells in a patient or subject.The term ‘prophylactic’ also encompasses the use of an agent, orformulation thereof, as described herein to prevent recurrence of aneoplastic disorder in a patient who has previously been treated for theneoplastic disorder.

In one embodiment, the neoplastic disorder is associated with theformation of solid tumours within the subject's body.

Thus, the solid tumour may be selected from the group consisting ofprostate cancer, breast cancer, lung cancer, colorectal cancer,melanomas, bladder cancer, brain/CNS cancer, cervical cancer,oesophageal cancer, gastric cancer, head/neck cancer, kidney cancer,liver cancer, lymphomas, ovarian cancer, pancreatic cancer and sarcomas.

For example, the solid tumour may be selected from the groups consistingof renal cell carcinoma, colorectal cancer, lung cancer, prostate cancerand breast cancer.

A ninth aspect of the invention provides a use of a bispecificpolypeptide according to the first aspect of the invention in thepreparation of a medicament for treating or preventing a neoplasticdisorder in a subject.

In one embodiment, the neoplastic disorder is associated with theformation of solid tumours within the subject's body (for example, asdetailed above).

A tenth aspect of the invention provides a method for the treatment ordiagnosis of a neoplastic disorder in a subject, comprising the step ofadministering to the subject an effective amount of a bispecificpolypeptide according to the first aspect of the invention.

In one embodiment, the neoplastic disorder is associated with theformation of solid tumours within the subject's body (for example, asdetailed above).

In one embodiment, the subject is human.

In one embodiment, the method comprises administering the bispecificantibody systemically.

In one embodiment, the methods further comprises administering to thesubject one or more additional therapeutic agents.

The listing or discussion of an apparently prior-published document inthis specification should not necessarily be taken as an acknowledgementthat the document is part of the state of the art or is common generalknowledge.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.”

These, and other, embodiments of the invention will be betterappreciated and understood when considered in conjunction with the abovedescription and the accompanying drawings. It should be understood,however, that the above description, while indicating variousembodiments of the invention and numerous specific details thereof, isgiven by way of illustration and not of limitation. Many substitutions,modifications, additions and/or rearrangements may be made within thescope of the invention without departing from the spirit thereof, andthe invention includes all such substitutions, modifications, additionsand/or rearrangements.

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a schematic representation of the structure of exemplaryformats for a bispecific antibody of the invention. In each format, theconstant regions are shown as filled light grey; variable heavy chainregions VH1 are shown as chequered black and white; variable light chainregions VL1 are shown as filled white; variable heavy chain regions VH2are shown as filled black; and variable light chain regions VL2 areshown as white with diagonal lines. CD137 binding domains (bindingdomain 1) are typically represented as a pair of a chequered black andwhite domain with a filled white domain (VH1/VL1); tumour-associatedantigen binding domains (binding domain 2) are typically represented asa pair of a filled black domain and a white domain with diagonal lines(VH2/VL2). However, in all of the formats shown, it will be appreciatedthat binding domains 1 and 2 may be switched. That is, a CD137 bindingdomain may occur in a position shown in this figure for atumour-associated antigen domain, and vice versa.

FIG. 2 shows an example of a dose-response experiment of 5T4 antibodiesbinding to 5T4-transfected B16 cells, analysed by flow cytometry.

FIG. 3 shows flow cytometry data showing normalized mean fluorescenceintensity (MFI) of 5T4 mAb binding at a concentration of 2.5 μg/ml to5T4-transfected B16 cells. The figure shows the mean±SD of the pooleddata from four experiments, with 1-4 data points for each antibody, asindicated in Table 2. MFI values were normalised to reference antibody1628.

FIG. 4 shows dose-response analysis of 5T4 antibody binding tocynomolgus 5T4-transfected CHO cells.

FIG. 5 is an illustration of 5T4 chimeras used for epitope mapping of5T4 antibodies. A: Each of the indicated domains E1-E7 were replaced bymouse 5T4 sequence in human/mouse chimeras. B: aa 173-420 were replacedby mouse 5T4 sequence

FIG. 6 shows binding of exemplary anti-CD137 antibodies to human andcynomolgus CD137. Data from two separate experiments are included.

FIG. 7 shows an overview of human/mouse CD137 chimeras. Black: mousesequence, white: human sequence.

FIG. 8 shows stimulation index values normalized to reference 1811/1812.

FIG. 9 shows the summary of two experiments of CD137 mAb competitionwith CD137L binding to CHO-huCD137 cells (25 μg/ml).

FIG. 10 shows CD137 activation in the presence of crosslinking antibody.

FIG. 11 shows CD137 activation in the absence of crosslinking antibody.

FIG. 12 shows dose-response curves in dual ELISA of 5T4-CD137 bispecificantibodies. Each graph includes data based on one 5T4 binder (1206 [i.e.1206/1207], 1208, 1210 or 1212) combined with various CD137 agonisticantibodies (1200 [i.e. 1200/1201], 1202, 1204, 1214, 1618, 1620 or1626).

FIG. 13 shows 5T4-dependent T cell activation by exemplary bispecificantibodies (bsAb) of the invention. Each bsAb (1 μg/ml) was run in CD8 Tcell assays based on 2-4 individual donors. The data is presented asmean fold change to reference (1200-1210) and error bars represent SD.The left part of the graph shows bispecific antibodies where the 5T4scFv has been fused to CD137 IgG, i.e. 1200-1206 etc, whereas the rightpart of the graph shows bispecific antibodies where the CD137 scFv hasbeen fused to 5T4 IgG, i.e. 1206-1200 etc.

FIG. 14 shows the dose-response of 5T4-CD137 bsAbs showing 5T4 dependentT cell activation. Data is analysed as fold change to reference(1200-1210 at 1 μg/ml). Upper panel: CD137 agonist as IgG and 5T4 binderas scFv fused to C-terminus of IgG. Lower panel: 5T4 binder as IgG andCD137 agonist as scFv fused to C-terminus of IgG. Clone designationfollows the same principle as described for FIG. 10.

FIG. 15 shows the functional activity of exemplary 5T4-CD137 bispecificantibodies on human CD8+ T cells cultured with 5T4-expressing tumorcells. All generated bsAbs were evaluated at 1 μg/ml in the fullycell-based T cell assay to verify the results obtained in the assayperformed with coated 5T4-Fc. Results are presented as fold change toreference (1200-1210) and the error bars are the SD. Clone designationfollows the same principle as described for FIG. 10.

FIG. 16 shows binding curves for 5T4 lead optimised clones to (A)CHOh5T4 and (B) CHOcyno5T4 cells.

FIG. 17 shows binding curves for CD137 lead optimised clones to (A)CHOhCD137 and (B) CHOcynoCD137 cells.

FIG. 18 shows the normalised interferon gamma (IFNγ) response in humanCD8+ T cells cultured in 5T4-Fc coated plates, represented as athree-parameter sigmoidal dose-response model to enable determination ofEC50.

FIG. 19 shows results for lead optimised bsAb in a CD8+ T cell assaywith crosslinked 5T4-Fc, with normalised IFNγ levels to enablecorrelation of results between assay plates.

FIG. 20 shows results for bsAbs generated with different linkers in aCD8+T cell assay with crosslinked 5T4-Fc, with normalised IFNγ levels toenable correlation of results between assay plates.

FIG. 21 shows the normalised interferon gamma (IFNγ) response using leadoptimised bsAb in human CD8+T cells cultured with 5T4-expressing and5T4-non-expressing tumour cells, represented as a three-parametersigmoidal dose-response model to enable determination of EC50

FIG. 22 shows the interferon gamma (IFNγ) response from CD137-mediatedactivation of PBMCs with and without the presence of 5T4-Fc.

FIG. 23 shows the interferon gamma (IFNγ) response from co-culture ofCD8+ T cells and CD32-expressing L cells.

FIG. 24 shows results from a dual ELISA detecting CD137, for TAA-CD137bispecific antibodies.

FIG. 25 shows the interferon gamma (IFNγ) response using TAA-CD137bispecific antibodies in CD8+ T cells cultured on CD3/TAA-coated plateswhere the TAA is (A) EpCAM, (B) EGFR and (C) Her2.

FIG. 26 shows 5T4-dependent localization of bispecific antibody to theantigen-expressing tumors. B16 and B16-5T4 tumors were collected fromSCID-Beige mice treated with vehicle, 1618-1210 (bsAb), 1618 (anti-CD137Mab) or 2112 (reference anti-CD137 Mab). Localization of antibody to thetumors was detected with anti-human IgG and analyzed by flow cytometry.The graph shows the frequency of human IgG+ cells among live cells(n=5).

FIG. 27 shows 5T4-dependent localization of bispecific antibody to theantigen-expressing tumors. CT26 and CT26-5T4 tumors were collected fromSCID-Beige mice treated with vehicle, 1618-1210, 1618 or 2112.Localization of antibody to the tumors was detected (A) with anti-humanIgG or (B) by binding of biotinylated CD137, and analyzed by flowcytometry. The graphs show the frequency of positive cells among single,live tumor cells (n=5).

FIG. 28 shows the percentage of tumor cells that are positive forbinding of biotinylated CD137 and the tumor antigen 5T4. SKOV-3 tumorswere collected from SCID-Beige mice treated with vehicle, 1618-1210,1618 or 2112. Localization of antibody to the 5T4 positive tumour cellswas detected with anti-human IgG and anti-human 5T4-antibody. The graphshow the frequency of double positive cells among single, live tumorcells (mCD45-CD45RA- (n=5/treatment).

TABLES (SEQUENCES)

TABLE A VL and VH amino acid (aa) and nucleotide (nt) sequences SEQ IDNO. CHAIN NO. TYPE SEQUENCE 1 1206, heavy aaEVQLLESGGGLVQPGGSLRLSCAASGFTFSGSSMSW chain, VHVRQAPGKGLEWVSSIYYSGSGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYGRNVHPYNLDY WGQGTLVTVSS 2 1206, heavy ntGAGGTGCAGCTGTTGGAGAGCGGGGGAGGCTTGGT chain, VHACAGCCTGGGGGGTCCCTGCGCCTCTCCTGTGCAGCCAGCGGATTCACCTTTTCTGGTTCTTCTATGTCTTG GGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCTATTTACTACTCTGGTTCTGGTACATACTATGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGTGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACGGCTGTATATTATTGTGCGCGCTACGGTCGTAACGTTCATCCGTACAACT TGGACTATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA 3 1207, light aa DIQMTQSPSSLSASVGDRVTITCRASOSISSYLNWYQQ chain VLKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISS LQPEDFATYYCQQGYYYLPTFGQGTKLEIK 41207, light nt GACATCCAGATGACCCAGTCTCCATCCTCCCTGAGC chain VLGCATCTGTAGGAGACCGCGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGAAGCGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTATTACTGTCAACAGGGTTACTACTACCTGCCCAC TTTTGGCCAGGGGACCAAGCTGGAGATCAAA 51208, heavy aa EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWV chain VHRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSPYYYGANWIDYW GQGTLVTVSS 6 1208, heavy ntGAGGTGCAGCTGTTGGAGAGCGGGGGAGGCTTGGT chain VHACAGCCTGGGGGGTCCCTGCGCCTCTCCTGTGCAG CCAGCGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTG GGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTATGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGTGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACGGCTGTATATTATTGTGCGCGCTCTCCGTACTACTACGGTGCTAACTGGATTGACTATTGGGGCCAGGGAACCCTGGTCACCGT CTCCTCA 7 1135, light aaDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQ chain VLKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISS LQPEDFATYYCQQSYSTPYTFGQGTKLEIK 81135, light nt GACATCCAGATGACCCAGTCTCCATCCTCCCTGAGC chain VLGCATCTGTAGGAGACCGCGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGAAGCGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTATTACTGTCAACAGAGTTACAGTACCCCTTATAC TTTTGGCCAGGGGACCAAGCTGGAGATCAAA 91210, heavy aa EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWV chain VHRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYYGGYYSAWMDY WGQGTLVTVSS 10 1210, heavy ntGAGGTGCAGCTGTTGGAGAGCGGGGGAGGCTTGGT chain VHACAGCCTGGGGGGTCCCTGCGCCTCTCCTGTGCAG CCAGCGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTG GGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTATGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGTGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACGGCTGTATATTATTGTGCGCGCTACTACGGTGGTTACTACTCTGCTTG GATGGACTATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA 11 1211, light aa DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQchain VL KPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTYGYLHTFGQGTKLEIK 12 1211, light ntGACATCCAGATGACCCAGTCTCCATCCTCCCTGAGC chain VLGCATCTGTAGGAGACCGCGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGAAGCGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTATTACTGTCAACAGACTTACGGTTACCTGCACAC TTTTGGCCAGGGGACCAAGCTGGAGATCAAA 131212, heavy aa EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWV chain VHRQAPGKGLEWVSYISSYGGYTSYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYHSGVLDYWGQG TLVTVSS 14 1212, heavy ntGAGGTGCAGCTGTTGGAGAGCGGGGGAGGCTTGGT chain VHACAGCCTGGGGGGTCCCTGCGCCTCTCCTGTGCAG CCAGCGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTG GGTCTCATACATTTCTTCTTACGGTGGTTACACATCTTATGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGTGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACGGCTGTATATTATTGTGCGCGCTACCATTCTGGTGTTTTGGACTATTGG GGCCAGGGAACCCTGGTCACCGTCTCCTCA 151213, light aa DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQ chain VLKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISS LQPEDFATYYCQQYYYHYLLTFGQGTKLEIK16 1213, light nt GACATCCAGATGACCCAGTCTCCATCCTCCCTGAGC chain VLGCATCTGTAGGAGACCGCGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGAAGCGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTATTACTGTCAACAGTACTACTACCATTACCTGCT CACTTTTGGCCAGGGGACCAAGCTGGAGATCAAA17 1200, heavy aa EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWV chain VHRQAPGKGLEWVSGISGGGGGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDVAYFDYWGQGTL VTVSS 18 1200, heavy ntGAGGTGCAGCTGTTGGAGAGCGGGGGAGGCTTGGT chain VHACAGCCTGGGGGGTCCCTGCGCCTCTCCTGTGCAG CCAGCGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTG GGTCTCAGGTATTTCTGGTGGTGGTGGTGGTACATACTATGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGTGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACGGCTGTATATTATTGTGCGCGCGACGTTGCTTACTTTGACTATTGGGG CCAGGGAACCCTGGTCACCGTCTCCTCA 191201, light aa DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQ chain VLKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISS LQPEDFATYYCQQYYIPHTFGQGTKLEIK 201201, light nt GACATCCAGATGACCCAGTCTCCATCCTCCCTGAGC chain VLGCATCTGTAGGAGACCGCGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGAAGCGGGACAGATTTCACTCTCACCATCAgCAGTCTGCAACCTGAAGAtTTTGCAACTTATTACTGTCAACAGTACTACATTCCGCACACTTTT GGCCaGGGGACCaAGCTGGagaTCAAA 211202, heavy aa EVQLLESGGGLVQPGGSLRLSCAASGFTFYGSSMSW chain VHVRQAPGKGLEWVSSIYYGSSGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSYYGYFDYWGQ GTLVTVSS 22 1202, heavy ntGAGGTGCAGCTGTTGGAGAGCGGGGGAGGCTTGGT chain VHACAGCCTGGGGGGTCCCTGCGCCTCTCCTGTGCAGCCAGCGGATTCACCTTTTACGGTTCTTCTATGTCTTG GGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCTATTTACTACGGTTCTTCTGGTACATACTATGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGTGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACGGCTGTATATTATTGTGCGCGCTCTTACTACGGTTACTTTGACTATTGGG GCCAGGGAACCCTGGTCACCGTCTCCTCA 231203, light aa DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQ chain VLKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISS LQPEDFATYYCQQYYTVVPFTFGQGTKLEIK24 1203, light nt GACATCCAGATGACCCAGTCTCCATCCTCCCTGAGC chain VLGCATCTGTAGGAGACCGCGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGAAGCGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTATTACTGTCAACAGTACTACACTGTTGTTCCGTT CACTTTTGGCCAGGGGACCAAGCTGGAGATCAAA25 1205, light aa DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQ chain VLKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISS LQPEDFATYYCQQSVPHYPFTFGQGTKLEIK26 1205, light nt GACATCCAGATGACCCAGTCTCCATCCTCCCTGAGC chain VLGCATCTGTAGGAGACCGCGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGAAGCGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTATTACTGTCAACAGTCTGTTCCGCACTACCCGTT CACTTTTGGCCAGGGGACCAAGCTGGAGATCAAA27 1204, heavy aa EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYYMGW chain VHVRQAPGKGLEWVSGIGSYYGYTGYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARAYYDYNYYYAYF DYWGQGTLVTVSS 28 1204, heavy ntGAGGTGCAGCTGTTGGAGAGCGGGGGAGGCTTGGT chain VHACAGCCTGGGGGGTCCCTGCGCCTCTCCTGTGCAGCCAGCGGATTCACCTTTTCTTCTTACTACATGGGTTG GGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGGTATTGGTTCTTACTACGGTTACACAGGTTATGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGTGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACGGCTGTATATTATTGTGCGCGCGCTTACTACGACTACAACTACTACTACGCTTACTTTGACTATTGGGGCCAGGGAACCCTGGTCA CCGTCTCCTCA 29 1214 (VH) aaEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIGSGGGYTGYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVGHPFDYWGQGT LVTVSS 30 1214 (VH) ntGAGGTGCAGCTGTTGGAGAGCGGGGGAGGCTTGGT ACAGCCTGGGGGGTCCCTGCGCCTCTCCTGTGCAGCCAGCGGATTCACCTTTAGCAGCTATGCCATGAGCT GGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCTATTGGTTCTGGTGGTGGTTACACAGGTTATGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGTGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACGGCTGTATATTATTGTGCGCGCGTTGGTCATCCGTTTGACTATTGGGG CCAGGGAACCCTGGTCACCGTCTCCTCA 311215 (VL) aa DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISS LQPEDFATYYCQQDAYPHTFGQGTKLEIK 321215 (VL) nt GACATCCAGATGACCCAGTCTCCATCCTCCCTGAGCGCATCTGTAGGAGACCGCGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGAAGCGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTATTACTGTCAACAGGACGCTTACCCGCACACTTT TGGCCAGGGGACCAAGCTGGAGATCAAA 331618 (VH) aa EVQLLESGGGLVQPGGSLRLSCAASGFTFSYGSMYWVRQAPGKGLEWVSSISSGSGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSSYYGSYYSIDY WGQGTLVTVSS 34 1618 (VH) ntGAGGTGCAGCTGTTGGAGAGCGGGGGAGGCTTGGT ACAGCCTGGGGGGTCCCTGCGCCTCTCCTGTGCAGCCAGCGGATTCACCTTTTCTTACGGTTCTATGTACTG GGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCTATTTCTTCTGGTTCTGGTTCTACATACTATGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCC GTGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACGGCTGTATATTATTGTGCGCGCTCTTCTTACTACGGTTCTTACTACTCTATTGACTATTGGGGCCAGGGAACCCTGGTCACCGTCTC CTCA 35 1619 (VL) aaDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISS LQPEDFATYYCQQYYDNLPTFGQGTKLEIK  36 1619 (VL) nt GACATCCAGATGACCCAGTCTCCATCCTCCCTGAGCGCATCTGTAGGAGACCGCGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGAAGCGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTATTACTGTCAACAGTACTACGACAACCTGCCCAC TTTTGGCCAGGGGACCAAGCTGGAGATCAAA 371620 (VH) aa EVQLLESGGGLVQPGGSLRLSCAASGFTFSGYYMYWVRQAPGKGLEWVSGISSSGSYTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSVGPYFDYWGQ GTLVTVSS 38 1620 (VH) ntGAGGTGCAGCTGTTGGAGAGCGGGGGAGGCTTGGT ACAGCCTGGGGGGTCCCTGCGCCTCTCCTGTGCAGCCAGCGGATTCACCTTTTCTGGTTACTACATGTACTG GGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGGTATTTCTTCTTCTGGTTCTTACACATACTATGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGTGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACGGCTGTATATTATTGTGCGCGCTCTGTTGGTCCGTACTTTGACTATTGGG GCCAGGGAACCCTGGTCACCGTCTCCTCA 391621 (VL) aa DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISS LQPEDFATYYCQQGVGPYTFGQGTKLEIK 401621 (VL) nt GACATCCAGATGACCCAGTCTCCATCCTCCCTGAGCGCATCTGTAGGAGACCGCGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGAAGCGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTATTACTGTCAACAGGGTGTTGGTCCGTACACTTT TGGCCAGGGGACCAAGCTGGAGATCAAA 411626 (VH) aa EVQLLESGGGLVQPGGSLRLSCAASGFTFGGYSMYWVRQAPGKGLEWVSSIGGYYYSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSYYGSIDYWGQG TLVTVSS 42 1626 (VH) ntGAGGTGCAGCTGTTGGAGAGCGGGGGAGGCTTGGT ACAGCCTGGGGGGTCCCTGCGCCTCTCCTGTGCAGCCAGCGGATTCACCTTTGGTGGTTACTCTATGTACT GGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCTATTGGTGGTTACTACTACTCTACATACTATGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGTGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACGGCTGTATATTATTGTGCGCGCTCTTACTACGGTTCTATTGACTATTGG GGCCAGGGAACCCTGGTCACCGTCTCCTCA 431627 (VL) aa DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISS LQPEDFATYYCQQGTGYGPLTFGQGTKLEIK44 1627 (VL) nt GACATCCAGATGACCCAGTCTCCATCCTCCCTGAGCGCATCTGTAGGAGACCGCGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGAAGCGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTATTACTGTCAACAGGGTACTGGTTACGGTCCGC TCACTTTTGGCCAGGGGACCAAGCTGGAGATCAAA

TABLE B 5T4 antibody sequences - CDR sequences Clone name (mAb) VH VL H1H2 H3 L1 L2 L3 1206/ 1206 1207 GFTFS IYYSGS ARYGR QSISSY AAS QQGYY 1207GSS GT NVHPY (SEQ ID (SEQ ID YLPT (SEQ ID (SEQ ID NLDY NO: 54) NO: 55)(SEQ ID NO: 45) NO: 47) (SEQ ID NO: 56) NO: 50) 1208/ 1208 1135 GFTFSSISGSGG ARSPY QSISSY AAS QQSYS 1135 YA (SEQ ST (SEQ YYGAN (SEQ ID (SEQ IDTPYT ID NO: ID NO: WIDY NO: 54) NO: 55) (SEQ ID 46) 48) (SEQ ID NO: 57)NO: 51) 1210/ 1210 1211 GFTFSS ISGSGG ARYYG QSISSY AAS QQTYG 1211YA (SEQ ST (SEQ GYYSA (SEQ ID (SEQ ID YLHT ID NO: ID NO: WMDY NO: 54)NO: 55) (SEQ ID 46) 48) (SEQ ID NO: 58) NO: 52) 1212/ 1212 1213 GFTFSSISSYGG ARYHS QSISSY AAS QQYYY 1213 YA (SEQ YT (SEQ GVLDY (SEQ ID (SEQ IDHYLLT ID NO: ID NO: (SEQ ID NO: 54) NO: 55) (SEQ ID 46) 49) NO: 53)NO: 59) 2992/ 2992 2993 GFDFE ISGSGG ARYYG QSIRSA AAS QQTYG 2993 SYAST (SEQ GYYSA (SEQ ID (SEQ ID YLHT (SEQ ID ID NO: WMDY NO: 145) NO:55)(SEQ ID NO: 144) 48) (SEQ ID NO: 58) NO: 52) 2994/ 2994 2995 GFDFDISGRGG ARYYG QSIRSA AAS QQTYG 2995 SYA ST (SEQ GYYSA (SEQ ID (SEQ IDYLHT (SEQ ID ID NO: WMDY NO: 145) NO:55) (SEQ ID NO: 146) 147) (SEQ IDNO: 58) NO: 52) 2996/ 2996 2997 GFDFD ISGSGG ARYYG QSIRQA AAS QQTYG 2997SYA ST (SEQ GYYSA (SEQ ID (SEQ ID YLHT (SEQ ID ID NO: WMDY NO: 148)NO: 55) (SEQ ID NO: 146) 48) (SEQ ID NO: 58) NO: 52) 2998/ 2998 2999GFDFD ISGSGG ARYYG QSISQA AAS QQTYG 2999 SYA ST (SEQ GYYSA (SEQ ID(SEQ ID YLHT (SEQ ID ID NO: WMDY NO: 149) NO: 55) (SEQ ID NO: 146) 48)(SEQ ID NO: 58) NO: 52) 3000/ 3000 3001 GFDFS ISGSGG ARYYG QSIRQA AADQQTYG 3001 SYA ST (SEQ GYYSA (SEQ ID (SEQ ID YLHT (SEQ ID ID NO: WMDYNO: 148) NO: 151) (SEQ ID NO: 150) 48) (SEQ ID NO: 58) NO: 52) 3002/3002 3003 GFTFDS ISGSGG ARYYG QSIRSA AAS QQTYG 3003 YA (SEQ ST (SEQGYYSA (SEQ ID (SEQ ID YLHT ID NO: ID NO: WMDY NO: 145) NO: 55) (SEQ ID152) 48) (SEQ ID NO: 58) NO: 52) 3004/ 3004 3005 GFDFD ISGSGG ARYYGQSISSA AAS QQTYG 3005 SYA ST (SEQ GYYSA (SEQ ID (SEQ ID YLHT (SEQ IDID NO: WMDY NO: 153) NO: 55) (SEQ ID NO: 146) 48) (SEQ ID NO: 58)NO: 52) 3006/ 3006 3007 GFDFE ISGSGG ARYYG QSIHQA GAS QQTYG 3007 SYAST (SEQ GYYSA (SEQ ID (SEQ ID YLHT (SEQ ID ID NO: WMDY NO: 154) NO: 155)(SEQ ID NO: 144) 48) (SEQ ID NO: 58) NO: 52) 3008/ 3008 3009 GFDFDISGSGG ARYYG QSIHQA AAS QQTYG 3009 SYA ST (SEQ GYYSA (SEQ ID (SEQ IDYLHT (SEQ ID ID NO: WMDY NO: 154) NO: 55) (SEQ ID NO: 146) 48) (SEQ IDNO: 58) NO: 52)

TABLE C CD137 antibodies - CDR sequences Table C(1) - VH Antibody CDRH1CDRH2 CDRH3 1200/1201 GFTFSSYA (SEQ ID ISGGGGGT (SEQ IDARDVAYFDY (SEQ ID NO: 46) NO: 65) NO: 72) 1202/1203 GFTFYGSS (SEQ IDIYYGSSGT (SEQ ID ARSYYGYFDY (SEQ ID NO: 60) NO: 66) NO: 73) 1204/1205GFTFSSYY (SEQ ID IGSYYGYT (SEQ ID ARAYYDYNYYYAYFDY NO: 61) NO: 67)(SEQ ID NO: 74) 1214/1215 GFTFSSYA (SEQ ID IGSGGGYT (SEQ IDARVGHPFDY (SEQ ID NO: 46) NO: 68) NO: 75) 1618/1619 GFTFSYGS (SEQ IDISSGSGST (SEQ ID ARSSYYGSYYSIDY NO: 62) NO: 69) (SEQ ID NO: 76)1620/1621 GFTFSGYY (SEQ ID ISSSGSYT (SEQ ID ARSVGPYFDY (SEQ ID NO: 63)NO: 70) NO: 77) 1626/1627 GFTFGGYS (SEQ ID IGGYYYST (SEQ IDARSYYGSIDY (SEQ ID NO: 64) NO: 71) NO: 78) 3012/3013 GFTFDYGS (SEQ IDISSGSGST (SEQ ID ARSSYYGSYYSIDY NO: 156) NO: 69) (SEQ ID NO: 76)3014/3015 GFTFSYGS (SEQ ID ISSGSGST (SEQ ID ARSSYYGSYYSIDY NO: 62)NO: 69) (SEQ ID NO: 76) 3016/3017 GFTFSYGS (SEQ ID ISSGSGST (SEQ IDARSSYYGSYYSIDY NO: 62) NO: 69) (SEQ ID NO: 76) 3018/3019GFTFDYGS (SEQ ID ISSGSGST (SEQ ID ARSSYYGSYYSIDY NO: 156) NO: 69)(SEQ ID NO: 76) 3020/3021 GFTFDYGS (SEQ ID ISSGSGST (SEQ IDARSSYYGSYYSIDY NO: 156) NO: 69) (SEQ ID NO: 76) 3022/3023GFTFDYGS (SEQ ID ISSGSGST (SEQ ID ARSSYYGSYYSIDY NO: 156) NO: 69)(SEQ ID NO: 76) 3024/3025 GFTFDYGS (SEQ ID ISSGSGST (SEQ IDARSSYYGSYYSIDY NO: 156) NO: 69) (SEQ ID NO: 76) 3026/3027GFTFDYGS (SEQ ID ISSGSGST (SEQ ID ARSSYYGSYYSIDY NO: 156) NO: 69)(SEQ ID NO: 76) 3028/3029 GFDFSYGS (SEQ ID ISSGSGST (SEQ IDARSSYYGSYYSIDY NO: 157) NO: 69) (SEQ ID NO: 76) 3030/3031GFTFDYGS (SEQ ID ISSGSGST (SEQ ID ARSSYYGSYYSIDY NO: 156) NO: 69)(SEQ ID NO: 76) 3032/3033 GFTFDYGS (SEQ ID ISSGSGST (SEQ IDARSSYYGSYYSIDY NO: 156) NO: 69) (SEQ ID NO: 76) 3034/3035GFTFSYGS (SEQ ID ISSGSGST (SEQ ID ARSSYYGSYYSIDY NO: 62) NO: 69)(SEQ ID NO: 76) 3036/3037 GFTFDYGS (SEQ ID ISSGSGST (SEQ IDARSSYYGSYYSIDY NO: 156) NO: 69) (SEQ ID NO: 76) Table C(2) - VL AntibodyCDRL1 CDRL2 CDRL3 1200/1201 QSISSY (SEQ ID NO: AAS (SEQ ID NO:QQYYIPHT (SEQ ID NO: 54) 55) 79) 1202/1203 QSISSY (SEQ ID NO:AAS (SEQ ID NO: QQYYTVVPFT (SEQ ID 54) 55) NO: 80) 1204/1205QSISSY (SEQ ID NO: AAS (SEQ ID NO: QQSVPHYPFT (SEQ ID 54) 55) NO: 81)1214/1215 QSISSY (SEQ ID NO: AAS (SEQ ID NO: QQDAYPHT (SEQ ID NO: 54)55) 82) 1618/1619 QSISSY (SEQ ID NO: AAS (SEQ ID NO:QQYYDNLPT (SEQ ID NO: 54) 55) 83) 1620/1621 QSISSY (SEQ ID NO:AAS (SEQ ID NO: QQGVGPYT (SEQ ID NO: 54) 55) 84) 1626/1627QSISSY (SEQ ID NO: AAS (SEQ ID NO: QQGTGYGPLT (SEQ ID 54) 55) NO: 85)3012/3013 QSISQY (SEQ ID NO: GAS (SEQ ID NO: QQYYDNLPT (SEQ ID NO: 158)155) 83) 3014/3015 QSIRQY (SEQ ID NO: SAD (SEQ ID NO:QQYYDNLPT (SEQ ID NO: 159) 160) 83) 3016/3017 QSIRQY (SEQ ID NO:GAS (SEQ ID NO: QQYYDNLPT (SEQ ID NO: 159) 155) 83) 3018/3019QSISQY (SEQ ID NO: SAE (SEQ ID NO: QQYYDNLPT (SEQ ID NO: 158) 161) 83)3020/3021 QSIRSY (SEQ ID NO: SAS (SEQ ID NO: QQYYDNLPT (SEQ ID NO: 162)163) 83) 3022/3023 QSIRQY (SEQ ID NO: GAS (SEQ ID NO:QQYYDNLPT (SEQ ID NO: 159) 155) 83) 3024/3025 QSISSY (SEQ ID NO:AAS (SEQ ID NO: QQYYDNLPT (SEQ ID NO: 54) 55) 83) 3026/3027QSIRSY (SEQ ID NO: GAD (SEQ ID NO: QQYYDNLPT (SEQ ID NO: 162) 165) 83)3028/3029 QSIRQY (SEQ ID NO: GAE (SEQ ID NO: QQYYDNLPT (SEQ ID NO: 159)166) 83) 3030/3031 QSISSY (SEQ ID NO: GAE (SEQ ID NO:QQYYDNLPT (SEQ ID NO: 54) 166) 83) 3032/3033 QSISSY (SEQ ID NO:AAS (SEQ ID NO : QQYYDNLPT (SEQ ID NO: 54) 55) 83) 3034/3035QSISSY (SEQ ID NO: GAS (SEQ ID NO: QQYYDNLPT (SEQ ID NO: 54) 155) 83)3036/3037 QSIRSY (SEQ ID NO: GAS (SEQ ID NO: QQYYDNLPT (SEQ ID NO: 162)155) 83)

Mutated IgG1 Antibody Sequence

IgG1 LALA-sequence (SEQ ID NO: 86)ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Linker Sequences

(SEQ ID NO: 87) SGGGGSGGGGS (SEQ ID NO: 88) SGGGGSGGGGSAP(SEQ ID NO: 89) NFSQP, (SEQ ID NO: 90) KRTVA (SEQ ID NO: 91) GGGSGGGG(SEQ ID NO: 92) GGGGSGGGGS (SEQ ID NO: 93) GGGGSGGGGSGGGGS

(SG)m, where m=1 to 7.

IgG Constant Region Sequences

IgG1 heavy chain constant region sequence: (SEQ ID NO: 94)ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIgG1 light chain constant region sequence: (SEQ ID NO: 95)RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC

TABLE D Lead optimised VH and VL amino acid sequences for CD137 and 5T4Table D(1) - 5T4-specific VH sequences (optimised sequences from“1210”; SEQ ID NO: 9) 2992 EVQLLESGGGLVQPGGSLRLSCAASGFDFESYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYYGGYYSAWMDYWGQGTLVTVSS (SEQ ID NO: 96) 2994EVQLLESGGGLVQPGGSLRLSCAASGFDFDSYAMSWVRQAPGKGLEWVSAISGRGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYYGGYYSAWMDYWGQGTLVTVSS (SEQ ID NO: 98) 2996EVQLLESGGGLVQPGGSLRLSCAASGFDFDSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYYGGYYSAWMDYWGQGTLVTVSS (SEQ ID NO: 100) 2998EVQLLESGGGLVQPGGSLRLSCAASGFDFDSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYYGGYYSAWMDYWGQGTLVTVSS (SEQ ID NO: 102) 3000EVQLLESGGGLVQPGGSLRLSCAASGFDFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYYGGYYSAWMDYWGQGTLVTVSS (SEQ ID NO: 104) 3002EVQLLESGGGLVQPGGSLRLSCAASGFTFDSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYYGGYYSAWMDYWGQGTLVTVSS (SEQ ID NO: 106) 3004EVQLLESGGGLVQPGGSLRLSCAASGFDFDSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYYGGYYSAWMDYWGQGTLVTVSS (SEQ ID NO: 108) 3006EVQLLESGGGLVQPGGSLRLSCAASGFDFESYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYYGGYYSAWMDYWGQGTLVTVSS (SEQ ID NO: 110) 3008EVQLLESGGGLVQPGGSLRLSCAASGFDFDSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYYGGYYSAWMDYWGQGTLVTVSS (SEQ ID NO: 112)Table D(2) - CD137-specific VH sequences (optimised sequences from“1618”; SEQ ID NO: 33) 3012EVQLLESGGGLVQPGGSLRLSCAASGFTFDYGSMYWVRQAPGKGLEWVSSISSGSGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSSYYGSYYSIDYWGQGTLVTVSS (SEQ ID NO: 114) 3014EVQLLESGGGLVQPGGSLRLSCAASGFTFSYGSMYWVRQAPGKGLEWVSSISSGSGSTHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSSYYGSYYSIDYWGQGTLVTVSS (SEQ ID NO: 116) 3016EVQLLESGGGLVQPGGSLRLSCAASGFTFSYGSMYWVRQAPGKGLEWVSSISSGSGSTHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSSYYGSYYSIDYWGQGTLVTVSS (SEQ ID NO: 118) 3018EVQLLESGGGLVQPGGSLRLSCAASGFTFDYGSMYWVRQAPGKGLEWVSSISSGSGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSSYYGSYYSIDYWGQGTLVTVSS (SEQ ID NO: 120) 3020EVQLLESGGGLVQPGGSLRLSCAASGFTFDYGSMYWVRQAPGKGLEWVSSISSGSGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSSYYGSYYSIDYWGQGTLVTVSS (SEQ ID NO: 122) 3022EVQLLESGGGLVQPGGSLRLSCAASGFTFDYGSMYWVRQAPGKGLEWVSSISSGSGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSSYYGSYYSIDYWGQGTLVTVSS (SEQ ID NO: 124) 3024EVQLLESGGGLVQPGGSLRLSCAASGFTFDYGSMYWVRQAPGKGLEWVSSISSGSGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSSYYGSYYSIDYWGQGTLVTVSS (SEQ ID NO: 126) 3026EVQLLESGGGLVQPGGSLRLSCAASGFTFDYGSMYWVRQAPGKGLEWVSSISSGSGSTHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSSYYGSYYSIDYWGQGTLVTVSS (SEQ ID NO: 128) 3028EVQLLESGGGLVQPGGSLRLSCAASGFDFSYGSMYWVRQAPGKGLEWVSSISSGSGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSSYYGSYYSIDYWGQGTLVTVSS (SEQ ID NO: 130) 3030EVQLLESGGGLVQPGGSLRLSCAASGFTFDYGSMYWVRQAPGKGLEWVSSISSGSGSTHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSSYYGSYYSIDYWGQGTLVTVSS (SEQ ID NO: 132) 3032EVQLLESGGGLVQPGGSLRLSCAASGFTFDYGSMYWVRQAPGKGLEWVSSISSGSGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSSYYGSYYSIDYWGQGTLVTVSS (SEQ ID NO: 134) 3034EVQLLESGGGLVQPGGSLRLSCAASGFTFSYGSMYWVRQAPGKGLEWVSSISSGSGSTHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSSYYGSYYSIDYWGQGTLVTVSS (SEQ ID NO: 136) 3036EVQLLESGGGLVQPGGSLRLSCAASGFTFDYGSMYWVRQAPGKGLEWVSSISSGSGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSSYYGSYYSIDYWGQGTLVTVSS (SEQ ID NO: 138)Table D(3) - 5T4-specific VL sequences (optimised sequences from“1211”; SEQ ID NO: 11) 2993DIQMTQSPSSLSASVGDRVTITCRASQSIRSALNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTYGYLHTFGQGTKLEIK (SEQ ID NO: 97) 2995DIQMTQSPSSLSASVGDRVTITCRASQSIRSALNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTYGYLHTFGQGTKLEIK (SEQ ID NO: 99) 2997DIQMTQSPSSLSASVGDRVTITCRASQSIRQALNWYQQKPGKAPKLLIYAASSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTYGYLHTFGQGTKLEIK (SEQ ID NO: 101) 2999DIQMTQSPSSLSASVGDRVTITCRASQSISQALNWYQQKPGKAPKLLIYAASSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTYGYLHTFGQGTKLEIK (SEQ ID NO: 103) 3001DIQMTQSPSSLSASVGDRVTITCRASQSIRQALNWYQQKPGKAPKLLIYAADSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTYGYLHTFGQGTKLEIK (SEQ ID NO: 105) 3003DIQMTQSPSSLSASVGDRVTITCRASQSIRSALNWYQQKPGKAPKLLIYAASSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTYGYLHTFGQGTKLEIK (SEQ ID NO: 107) 3005DIQMTQSPSSLSASVGDRVTITCRASQSISSALNWYQQKPGKAPKLLIYAASSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTYGYLHTFGQGTKLEIK (SEQ ID NO: 109) 3007DIQMTQSPSSLSASVGDRVTITCRASQSIHQALNWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTYGYLHTFGQGTKLEIK (SEQ ID NO: 111) 3009DIQMTQSPSSLSASVGDRVTITCRASQSIHQALNWYQQKPGKAPKLLIYAASSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTYGYLHTFGQGTKLEIK (SEQ ID NO: 113)Table D(4) - CD137 specific VL sequences (optimised sequences from“1619 ”; SEQ ID NO: 35) 3013DIQMTQSPSSLSASVGDRVTITCRASQSISQYLNWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYDNLPTFGQGTKLEIK (SEQ ID NO: 115) 3015DIQMTQSPSSLSASVGDRVTITCRASQSIRQYLNWYQQKPGKAPKLLIYSADSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYDNLPTFGQGTKLEIK (SEQ ID NO: 117) 3017DIQMTQSPSSLSASVGDRVTITCRASQSIRQYLNWYQQKPGKAPKLLIYGASSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYDNLPTFGQGTKLEIK (SEQ ID NO: 119) 3019DIQMTQSPSSLSASVGDRVTITCRASQSISQYLNWYQQKPGKAPKLLIYSAESLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYDNLPTFGQGTKLEIK (SEQ ID NO: 121) 3021DIQMTQSPSSLSASVGDRVTITCRASQSIRSYLNWYQQKPGKAPKLLIYSASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYDNLPTFGQGTKLEIK (SEQ ID NO: 123) 3023DIQMTQSPSSLSASVGDRVTITCRASQSIRQYLNWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYDNLPTFGQGTKLEIK (SEQ ID NO: 125) 3025DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYDNLPTFGQGTKLEIK (SEQ ID NO: 127) 3027DIQMTQSPSSLSASVGDRVTITCRASQSIRSYLNWYQQKPGKAPKLLIYGADSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYDNLPTFGQGTKLEIK (SEQ ID NO: 129) 3029DIQMTQSPSSLSASVGDRVTITCRASQSIRQYLNWYQQKPGKAPKLLIYGAESLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYDNLPTFGQGTKLEIK (SEQ ID NO: 131) 3031DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYGAESLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYDNLPTFGQGTKLEIK (SEQ ID NO: 133) 3033DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYDNLPTFGQGTKLEIK (SEQ ID NO: 135) 3035DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYGASSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYDNLPTFGQGTKLEIK (SEQ ID NO: 137) 3037DIQMTQSPSSLSASVGDRVTITCRASQSIRSYLNWYQQKPGKAPKLLIYGASSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYDNLPTFGQGTKLEIK (SEQ ID NO: 139) Table D(5) - Connector sequencesReference Amino acid sequence SEQ ID m6 GGGGSGGGGS SEQ ID NO: 92 m15THTCPPCPEPKSSDK SEQ ID NO: 140 m16 GGGGS SEQ ID NO: 141 m17EAAKEAAKGGGGS SEQ ID NO: 142 m18 EAAKEAAK SEQ ID NO: 143Table D(6) - Additional alterations (modifications) Reference Alterationm2 L234A, L235A Fc mutations m5G49C in heavy chain and Q120C in light chain of scFv m19P15G, G16N, G17E, S18T in heavy chain of scFv

TABLE E Example describing how to translate the Antibody name into afull IgG sequence for bispecific antibodies in Morrison formatComposition of construct A B C D Antibody (VH (VL (VH (VL Con-Additional name of B1) of B1) of B2) of B2) nector* alterations* 1618-1618 1619 2992 2993 m6 m2 1210LO1 *See Table D(5) and D(6) above fordetails

Heavy chain: [A (underlined); Heavy chain Fc sequence withmodification m2; connector m6 (italic); C (bold);linker; D (bold underlined)] [SEQ ID NO: 167]EVQLLESGGGLVQPGGSLRLSCAASGFTFSYGSMYWVRQAPGKGLEWVSSISSGSGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSSYYGSYYSIDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG KGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFDFESYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYYGGYYSAWMDYWGQGTLVTVSSGGGGSGGGGSGGGGS DIQMTQSPSSLSASVGDRVTITCRASQSIRSALNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTYGYLHTFGQGTK LEIK Light chain:[B (bold underlined); Light chain constant sequence] [SEQ ID NO: 168]DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYDNLPTFGQ GTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC

EXAMPLES Example 1 Selection of 5T4 Antibodies from Alligator-GOLD™Library

Phage display selections against h5T4 were performed using the scFvlibrary ALLIGATOR-GOLD™, a fully human scFv library containing more than1×10¹⁰ unique members (Alligator Bioscience AB, Lund, Sweden). Severaldifferent selection strategies were employed, including solid phaseselection, selection in solution using biotinylated 5T4-Fc, selectionwith biotinylated 5T4-Fc coupled to streptavidin beads as well as oneround of selection against 5T4 expressing B16 cells using a phage stockthat previously had been selected against the recombinant h5T4-Fc. Priorto selection, phage stocks were pre-selected against streptavidin,Beriglobin or SLIT2 in order to remove potential binders tostreptavidin, the Fc part of the target and binders cross reactive toother leucine rich repeat proteins.

To identify specific binders from the phage selection, approximately1250 individual clones were screened in phage format using ELISA coatedwith 5T4-Fc or non-target protein (Biglycan or Orencia). This wasfollowed by sequence analysis as well as screening as soluble scFv infull-curve ELISA, ELISA performed at 50° C. and FACS analysis ofselected clones. Based on this, 14 unique candidate scFv were chosenwhich bound to recombinant 5T4 and to 5T4 expressing cells withoutshowing positive response to non-target molecules or to 5T4 negativecells.

The selected 14 5T4 scFv clones were converted to full IgG1 for furthercharacterization. A reference anti-5T4 antibody, designated 1628(selected from a representative prior art disclosure), was used in thisstudy as a positive control.

Among the 14 clones, four clones were selected for further evaluation inbispecific antibody format. These four clones are described furtherbelow, and compared to the reference clone 1628.

Example 2 Binding to Human 5T4 Measured by ELISA

Materials and Methods

ELISA was performed using a standard protocol. Plates (#655074, GreinerBio-One GmbH, Germany) were pre-coated with 0.5 μg/ml 5T4-Fc (obtainedfrom Peter L. Stern, University of Manchester) overnight. 5T4 antibodieswere diluted from 6 to 1.5×10⁻³ μg/ml in 1:4 dilutions and added induplicates of 50 μl to each well. Binding was detected with rabbitanti-h kappa L-chain-HRP (P0129, Dako Denmark) and the ELISA wasdeveloped with SuperSignal ELISA PICO Chemiluminescent substrate (ThermoScientific Pierce, Rockford, Ill. USA) for 2-10 minutes and read in anautomated microplate based multi-detection reader (FLUOstar OPTIMA,Netherlands).

Results and Conclusions

The results show that the majority of the 5T4 mAbs bind with similarpotency to 5T4 as 1628 (Table 1) with EC50 values in the sub-nM range.However, clone 1208 exhibits a slightly higher EC50 value.

TABLE 1 Summary of the obtained EC50 in the ELISA of all 5T4 mAb withthe confidence intervals and number of experiments EC50 (nM) Mean 95%EC50 Confidence Clone name (nM) Intervals n Reference 1628 0.56 0.3-1.08 1206 0.64 0.3-1.4 4 1208 2.24 2.0-2.4 1 1210 0.48 0.2-1.1 4 1212 0.560.2-1.2 4

Example 3 Binding to 5T4 expressed on the cell surface determined byflow cytometry

Materials and Methods

Analysis of 5T4 mAb binding with flow cytometry was performed using5T4-transfected cell lines and as negative control, mock transfectedcells. Three different transfected cell lines used were used for thisstudy; B16, A9 and CHO, transfected either with a 5T4 construct or withan empty vector control construct. Cells were stained with 5T4antibodies diluted in FACS buffer (PBS, 0.5% BSA and 0.02% NaN₃).Binding was detected with the secondary antibody anti-IgG (Fc)-PE(109-115-098, Jackson ImmunoResearch Europe, UK) diluted 1:100. Sampleswere analysed either on a FACSCalibur or a FACSverse (BD Biosciences,Heidelberg, Germany) and mean fluorescence intensity (MFI) determined.

Results and Conclusions

In flow cytometric analysis of 5T4 antibody binding to 5T4-transfectedB16 cells, most antibodies show good binding. Large variations in EC50values between individual experiments were observed. Therefore, resultsare summarized as mean EC50 in nM as well as mean EC50 normalized to theinternal control 1628 (Table 2). An example of dose-response curves forbinding of 5T4 mAb to 5T4-transfected B16 cells is shown in FIG. 2. InFIG. 3, normalized MFI values at a fixed concentration of 2.5 μg/mlantibody is shown. Taken together, the data indicate that mostantibodies bind well to 5T4-transfected B 16 cells, with clone1208exhibiting weaker binding.

TABLE 2 Potency of 5T4 antibodies as determined by flow cytometricanalysis of 5T4-transfected B16 cells EC50 (nM) Normalized EC50* CloneMean SD Mean SD n 1206 1.8 1.6 3.9 2.7 4 1208 0.7 9.3 1 1210 0.8 0.7 1.61.3 4 1212 1.4 2.2 1.1 0.3 4 Reference 1628 1.1 1.4 1.0 4 *EC50 valuenormalized to 1628

In a new attempt to calculate EC50 with flow cytometry a 5T4 mAb doseresponse experiment was performed using CHO cells stably transfectedwith human 5T4. A one to four titration series was performed startingfrom 2.5 nM. The data are summarized in Table 3.

TABLE 3 Summary of EC50 values, EC50 95% confidence intervals and EC50normalised to 1628 in flow cytometric analysis of 5T4-transfected CHOcells. Data was normalised and the EC50 values were calculated bynonlinear regression. 1206 1208 1210 1628 EC50 nM 0.51 2.07 0.81 0.51EC50 0.2 to 1.2 1.6.to 2.7 0.3 to 2.2 0.14 to 1.8 (95% confidenceintervals) Normalized to 1.0 4.1 1.6 1.0 reference 1628

Finally, binding potency to 5T4-transfected A9 cells was evaluated intwo individual experiments. As in the experiments performed with B16-5T4cells, the absolute EC50 values determined in individual experimentsvary, and data is therefore presented as normalized to the reference1628 (Table 4). Results indicate that the 5T4 antibodies bind withcomparable potency to the reference 1628.

TABLE 4 Potency of 5T4 antibodies as determined by flow cytometricanalysis of 5T4-transfected A9 cells Normalized EC50* Clone Mean SD n1206 2.9 1.9 2 1208 3.9 2.2 2 1210 1.8 0.2 2 1212 0.4 — 1 1628 1.0 — 2*EC50 value normalized to reference 1628

To summarize, the binding potency of four 5T4 antibodies was evaluatedby flow cytometry using three different 5T4-transfected cell lines (B16,CHO and A9). The conclusion from these studies is that all antibodiesexhibit reasonable binding, with clone 1208 in general exhibiting lowerpotency than the other clones.

Example 4 Binding to Cynomolgus 5T4

Materials and Methods

The potency of 5T4 antibodies in binding to cynomolgus 5T4 wasdetermined by flow cytometry. CHO cells were stably transfected withMacaca mulatta (cynomolgus) 5T4. Cells were stained with 5T4 antibodiesdiluted in FACS buffer (PBS, 0.5% BSA and 0.02% NaN₃) using a 1:4titration starting at 2.5 nM. Binding was detected with the secondaryantibody anti-IgG (Fc)-PE (109-115-098, Jackson ImmunoResearch Europe,UK) diluted 1:100. Samples were analysed either on a FACSCalibur or aFACSverse (BD Biosciences, Heidelberg, Germany) and mean fluorescenceintensity (MFI) determined. Three experiments were performed withcomparable results, although only one experiment included a fulldose-response curve whereas the other two experiments included onlythree antibody concentrations. To compare the EC50 values between humanand cynomolgus 5T4, the cy5T4/hu5T4 ratio was calculated from theexperiment with the full dose-response.

Results and Conclusions

The three experiments that were performed demonstrate good binding tocynomolgus 5T4 by clones 1206, 1208 and 1210 and weak binding by 1212and the reference 1628 (FIG. 4, Table 5) Clone 1206 had a relativelygood potency, but low efficacy. Comparison of the relative EC50 valuesbetween cynomolgus and human 5T4 for selected clones shows that clones1206, 1208 and 1210 have a relatively high affinity for cynomolgus 5T4whereas 1212 does not.

TABLE 5 EC50 values for cyno5T4 transfected cells and EC50 95%confidence intervals and the EC50cyno:EC50 human Antibody 1206 1208 12101212 1628 EC50 nM 1.53 0.96 0.70 30.7 93.0 EC50 1.1 to 2.1 0.5 to 1.80.3 to 1.7 21 to 45 37 to 235 (95% confidence intervals) RatioEC50cyno5T4/h5T4 3.0 0.5 0.9 140 182 Data were normalised and the EC50values were calculated by nonlinear regression

Example 5 Affinity determined by surface plasmon resonance

Materials and Methods

Binding kinetics of the 5T4-specific mAbs have been studied using twodifferent SPR-based platforms, the Biacore 3000 (GE Healthcare) and theMASS-1 platform (Sierra Sensors). Briefly, 5T4 was captured at thesensor chip surface either via direct amine coupling (Biacore platform)or using a streptavidin coated chip and biotinylated 5T4 (MASS-1platform). The different 5T4-specific mAbs were then injected over thechip in increasing concentrations and the association and dissociationrates studied in real time. A 1:1 Langmuir model was used for curvefitting.

Results and Conclusions

A summary of binding rate constants and affinities obtained using thetwo platforms is presented in Table 6. It should however be taken intoconsideration that the assay setup used allows for bivalent binding ofthe mAbs to the antigen. This will give rise to avidity effects thatlead to a significant underestimation of the off-rates (kd) and thusalso the affinity value (KD). The different 5T4 antibodies showdifferent binding characteristics, with 1208 and the reference 1628displaying very low off-rates while on-rates vary less between thebinders. It is obvious that there are significant variations between thetwo assays, with an over 10-fold difference for 1206 and 1628. For 1628this is likely due the difficulty in accurate curve fitting when theoff-rate becomes very low (close to no dissociation).

TABLE 6 Summary of binding kinetics of 5T4-specific mAbs Biacore MASS-1Clone ka (1/Ms) kd (1/s) KD (M) ka (1/Ms) kd (1/s) KD (nM) 1206 1.3E+052.8E−04 2.3E−09 1.4E+06 1.3E−04 9.7E−11 1208 — — — 2.1E+05 2.1E−069.8E−12 1210 5.0E+05 1.0E−04 2.0E−10 5.1E+05 1.9E−04 3.7E−10 12124.5E+05 8.1E−04 1.8E−10 — — — 1628 6.4E+05 2.6E−08 4.1E−14 1.5E+062.1E−06 1.5E−12

Example 6 Domain mapping of 5T4 antibodies

Materials and Methods

Epitope mapping was performed by investigation of loss of binding by theantibodies using a panel of human/mouse chimeric 5T4 constructs by flowcytometry. This strategy was possible since none of the 5T4 antibodiescross-react with murine 5T4. Two strategies were used for the epitopemapping as illustrated in FIG. 5. In one approach, seven human/mouse 5T4chimeras were constructed based on dividing 5T4 into seven differentdomains (FIG. 5). By replacing each domain with the corresponding mousesequence seven human/mouse 7 5T4 human/mouse chimeras were generated.The chimeras were generated using the human protein 5T4 sequenceNP_006661.1 (reference mRNA sequence NM_006670.4) and the correspondingmouse sequence NP_035757.2 (reference mRNA sequence NM_011627.4). Thehuman/mouse chimeric DNA constructs, as well as human and mousewild-type 5T4, were cloned into pcDNA3.1 expression vectors. Stablytransfected CHO cells were generated and 5T4 expressing cells enrichedby MACS sorting, resulting in 60-80% positive cells. In the otherapproach, cells transfected with a human/mouse 5T4 chimera (Woods etal., 2002, Biochem J 366(1):353-365) was used, in which mouse sequencein amino acid 173-420 replaced the human sequence (FIG. 5). As controlshuman 5T4 and mouse 5T4-transfected cells were used. For flow cytometricanalysis, cells were stained with different 5T4 antibodies diluted inFACS buffer (PBS, 0.5% BSA). Binding was detected with the secondaryantibody anti-IgG (Fc)-PE (109-115-098, Jackson ImmunoResearch Europe,UK) diluted 1:100. Samples were analysed by FACSverse (BD Biosciences,Heidelberg, Germany) and % positive cells were determined. To compensatefor variations in % 5T4 positive cells in the various transfectedpopulations, binding levels were normalized within each chimera bydividing % positive cells for each clone with % positive cells for theclones resulting in the highest % positive cells (% pos cells_(cloneX)/%positive cells_(max)). A normalized value ≤0.75 was defined as mAbbinding being dependent on the replaced region, whereas a normalizedvalue ≤0.25 was defined as complete dependence.

Results and Conclusion

The four 5T4 antibodies were shown to be more or less dependent on atleast one of domains E2, E3, E4, E6 or aa 173-420, whereas no cleardependence on E1, E5 or E7 was observed (Table 7).

All four antibodies had a distinct binding pattern:

-   -   1. Clone 1208; dependent on E2 and E4    -   2. Clone 1210; dependent on E2, E4 and aa173-420    -   3. Clone 1206; dependent on E2, E3, E4 and aa173-420    -   4. Clone 1212 dependent on E6 aa173-420

The reference antibody 1628 differed from all the exemplary antibodiesof the invention, and was completely dependent on E4 and aa173-420.

TABLE 7 Summary of epitope mapping results summarized as normalizedvalues for one representative experiment. Clone Group E1 E2 E3 E4 E5 E6E7 aa173-420 1628 0.96 1.00 1.00 0.06 0.96 0.81 0.91 0.00 1208 1 0.950.68 0.96 0.65 1.00 0.94 0.99 1.00 1210 2 0.99 0.02 0.90 0.69 0.89 0.890.96 0.00 1206 3 0.96 0.74 0.25 0.52 0.90 0.94 0.94 0.30 1212 4 0.890.90 0.93 1.00 0.84 0.03 0.88 −0.01 The experiment was repeated once forE2, E3 and E6 chimeras and three times for the E4 chimera with highreproducibility. mAbs with a normalized binding value ≤0.75 areindicated in bold.

Example 7 Selection of CD137 Antibodies from Alligator GOLD® Library

Phage display selections were performed using a human antibody (scFv)library, Alligator GOLD® (Alligator Bioscience, Lund, Sweden).Selections towards recombinant CD137 in soluble form, coated onto thesurface of beads or tubes, or expressed on the surface ofCD137-transfected cells were performed. CTLA4-Fc and an irrelevantHis-tagged protein were used as non-targets included in excess in theselections. Prior to each selection round, the phage stocks werepre-selected towards biotinylated beriglobin, CTLA4-Fc, beads or CD137negative cells to remove unspecific binders.

To identify specific binders from the phage selection, approximately4500 individual clones were screened in phage format using ELISA coatedwith either recombinant target (CD137-Fc) or non-target (CTLA4-Fc)protein, followed by confirmation as soluble scFv for some clones.Clones exhibiting specific binding to CD137 were sequenced and uniqueclones were produced as IgG for further characterization.

Example 8 Binding to Human CD137 Measured by ELISA

Material and Methods

Binding of CD137 antibodies to recombinant human CD137 was determined bysandwich ELISA. Briefly, ELISA plates (Greiner #655074) coated withrecombinant human CD137-Fc (R&D #838-4B) were incubated with serialdilutions of the various CD137 antibodies to be investigated. CD137antibodies were detected using HRP-conjugated goat-anti-human kappalight chain (AbD Serotec #STAR127P) and developed with SuperSignal ELISAPico Chemiluminescent substrate (Pierce #37069). EC50 values of thevarious antibodies were determined in 2-6 separate experiments.

Two different reference anti-CD137 antibodies have been used in thisstudy, as positive controls (designated “1811/1812” and “1813/1814”,both of which are available in the art).

Results and Conclusion

The majority of the antibodies exhibit EC50 values in a similar range asthose of the reference antibodies, i.e. sub nM or low nM. Data aresummarized in Table 8.

TABLE 8 EC50 values (nM) of Alligator-GOLD-derived CD137 antibodiesdetermined by ELISA for human CD137 Clone name Mean SD n 1811/1812 0.750.137 8 1813/1814 0.33 0.069 5 1200/1201 0.39 0.037 3 1202/1203 0.410.050 4 1204/1205 0.34 0.058 6 1214/1215 0.98 0.124 6 1618/1619 0.350.018 4 1620/1621 0.38 0.137 2 1626/1627 0.22 0.057 2 n = number of datapoints.

Example 9 Flow Cytometric Determination of Binding to Human andCynomolgus CD137

Material and Methods

Binding and EC50 was determined using flow cytometric analysis of CHOcells transfected with human CD137, cynomolgus CD137 or empty vector.The extracellular part of human or cynomolgus CD137 was fused to thetransmembrane and intracellular part of human CD40 and cloned intopcDNA3.1. The vector was subsequently stably transfected into CHO cells.Expression of CD137 was confirmed by flow cytometry using CD137 antibody(human CD137-PE, BD Biosciences #555956) for 30 min at 4° C.CD137-transfected and empty vector-transfected cells were incubated withCD137 antibodies for at least 1h at 4° C. to saturate the binding. Inorder to minimize antibody internalization, 0.05% sodium azide was usedin the incubation buffer and all work was performed on ice. The CD137antibodies were detected using PE-conjugated anti-hIgG antibody(109-115-098, Jackson Immunoresearch laboratories), incubated for 30 minat 4° C. Directly after staining the cells were fixed with aparaformaldehyde solution (10x concentrate BD CellFIX, BD biosciences#340181). Cells were analyzed by flow cytometry using FACSVerse (BDBiosciences). The median fluorescence intensity (MFI) for each samplewas determined and the dose response data was analysed using Graph PadPrism.

MFI data was normalized for each antibody, where 0% is defined as thelowest value and 100% is the highest value in the dose titration foreach antibody. EC50 and 95% confidence interval were calculated withGraph Pad Prism based on data from the two experiments (non-linearregression (curve fit), constraints set to 0 and 100).

Results and Conclusion

Binding to CHO-huCD137, CHO-cyCD137 and CHO-pcDNA was confirmed in twoseparate experiments (FIG. 6). All CD137 antibodies bind relatively wellto human CD137 with EC50 comparable with the two reference antibodies1811/1812 and 1813/1814. The majority of the CD137 antibodies testedbind well to cynomolgus CD137, except for reference antibody 1811/1812and 1200/1201 (data not shown) which do not bind at all or very weakly,and clone 1620/1621 which binds weakly and does not reach a completesaturation. It should be noted that the maximum MFI obtained on thecynomolgus CD137 cells were 2-3 fold lower than on the human CD137expressing cells, which indicate differences in receptor density on thecells.

The EC50 determination is presented as 95% confidence intervals for eachCD137 antibody tested in order to include the inter and intra assayvariations (Table 9).

TABLE 9 95% confidence intervals for the EC50 of each CD137 antibodydetermined as an average from two experiments of normalized data Bindingto human Binding to cyno CD137, CD137, Ratio, Clone name EC50 (μg/mL)EC50 (μg/mL) cyno:human 1811/1812 1.00-1.99 Nd Nd 1813/1814 0.21-0.31 0.13-0.24  0.69 1200/1201 0.20-0.36 Nd Nd 1202/1203 0.16-0.27 0.11-0.17  0.67 1204/1205 0.23-0.39  0.11-0.16  0.43 1214/12150.89-1.28  0.41-0.80  0.54 1618/1619 0.11-0.19 0.086-0.15  0.771620/1621 0.20-0.42    3-5* 14* 1626/1627 0.38-0.67  0.16-0.27  0.41*The estimated 95% confidence interval is likely underestimated Nd: nodata due to incomplete binding to target.

Example 10 Affinity of CD137 Antibodies Measured by Biacore

Material and Methods

Human CD137 (R&D systems) was immobilized to the Biacore™ sensor chip,CMS, using conventional amine coupling. The tested antibody and control(serially diluted 1/2 10-0.63 nM) were analyzed for binding in HBS-P(GE, #BR-1003-68) at a flow rate of 30 μl/ml. The association wasfollowed for 5 minutes and the dissociation for 15 minutes. Regenerationwas performed twice using 10 mM Glycine pH 1.7 for 30 seconds. Thekinetic parameters and the affinity constants were calculated using 1:1Langmuir model.

Results and Conclusion

The affinities of the antibodies were in the nanomolar to sub-nanomolarrange (Table 10) measured using bivalent antibodies flowed over CD137coated on the chip surface.

TABLE 10 Kinetic parameters measured by surface plasmon resonance Sampleka (1/Ms) kd (1/s) KD (M) 1200 ND ND ND 1202 6.76E+05 6.60E−04 9.76E−101204 2.54E+05 2.80E−04 1.10E−09 1214 4.54E+04 3.17E−05 6.99E−10 16181.02E+06 1.10E−04 1.07E−10 1620 3.92E+05 5.19E−04 1.32E−09 1626 2.32E+052.94E−04 1.27E−09 1814 1.05E+06 4.45E−04 4.24E−10 ND; not determined

Example 11 Target specificity of the CD137 antibodies determined byELISA

Material and Methods

Binding to TNFR superfamily members for which ELISA methods had alreadybeen established (CD40 and OX40) was evaluated to detect potentialpropensity to cross react to non-target proteins. In addition, a BLASTsearch was performed identifying TNFRSF21 as the most similar sequence(34% sequence identity). Since this sequence similarity is rather low,determination of non-target binding to OX40 and CD40 was consideredsufficient.

ELISA plates (Greiner #655074) were coated with 50 μl/well ofrecombinant human OX40 (R&D #1493-CD), CD40-Fc (Ancell #504-820) orCD137 (R&D #838-4B) diluted to a final concentration of 0.5 μg/ml in PBSfor 1h at 37° C. or overnight at 4° C. Plates were washed with PBS+0.05%TWEEN20 (PBST), followed by block with PBST+1% bovine serum albumin(BSA). Antibody samples were prepared as serial 1/10 dilutions from10-0.01 μg/ml in PBST+1% BSA and incubated for 1 h in room temperature,followed by detection using a horse radish peroxidase-conjugatedanti-human kappa light chain antibody (AbD Serotec #STAR127P) anddeveloped using SuperSignal ELISA Pico Chemiluminescent substrate(Pierce ThermoScientific #37069).

Results and Conclusion

The results from the two experiments were similar. One antibody(1202/1203) exhibited weak binding to OX40 and CD40, whereas none of theremaining antibody showed any detectable binding to either OX40 or CD40.An overview of antibodies analyzed, and results from the two experimentsis shown in Table 11. The EC50 for 1202/1203 binding to CD137 in ELISAwas determined as 0.41 nM, corresponding to approx. 0.06 μg/ml. TheELISA signal is very low even at 10 μg/ml, and the EC50 for binding toOX40 and CD40 is most likely higher than 10 μg/ml since the dilutioncurves have not reached a plateau. Further, binding to primary PBL frommultiple blood donors was tested. The binding to PBL was similar toReference antibodies. No relevant unspecific binding to non-targetproteins was detected.

TABLE 11 Summary of CD137 antibody unspecific binding to OX40 and CD40pAb Binding to OX40 and CD40 EC50 CD137 1200/1201 No 1202/1203 Weak;EC50 > 40 nM 0.4 nM 1204/1205 No 1214/1215 No 1618/1619 No 1620/1621 No1626/1627 No

Example 12 Domain Mapping of Antibodies Binding to CD137

Material and Methods

The ability of each antibody to bind to a panel of human/mouse CD137chimeras expressed on the surface of transfected cells was analyzed byflow cytometry.

The chimeras were designed by exchanging domains or modules of the humanCD137 with the corresponding mouse domain (FIG. 7). Genes of CD137human/mouse chimeras were synthesized (GenScript) and constructs clonedinto pcDNA3.1 vector (Invitrogen) and transiently transfected intoFreeStyle 293-F cells (Invitrogen). The transfected cells were incubatedwith CD137 antibodies and control antibodies, followed by incubationwith anti-human IgG-PE (Jackson Immunoresearch) for detection andanalyzed with FACS Verse (BD Biosciences). Binding to the differentchimeric constructs was calculated as relative MFI compared to thebinding of the isotype control, followed by normalization to thefull-length human CD137 construct to minimize the effect of affinitydifferences between individual antibodies.

Results and Conclusion

Four binding patterns can be observed as described below. Data issummarized in Table 12.

Pattern A:

Antibodies 1811/1812 (Reference antibody) and 1618/1619 are dependent ondomain 1.

Pattern B:

Antibodies 1200/1201, 1202/1203 and 1204/1205 are mainly dependent ondomain 2. In addition, some loss of binding is also seen for construct1555, indicating an impact of domain 1 as well.

Pattern C:

Antibodies 1813/1814 (Reference antibody) and 1620/1621 appear to bemainly dependent on domains 3B-4A. However, loss of binding is seen forall constructs, making this pattern quite similar to pattern D.

Pattern D:

For antibodies 1214/1215 and 1626/1627, no clear dependence onparticular CD137 domains could be demonstrated. Instead, theseantibodies exhibited extensive loss of binding for all chimeras.However, for 1214/1215, the results differed between the experiments(see Table 12).

TABLE 12 Median fluorescence intensity (MFI) for antibody sample/isotypecontrol, normalized to full-length human CD137 Group A B C D Domain 1 23B-4A Unclear Clone 1214 1811 1618 1200 1202 1204 1813 1620 1626 12151812 1619 1201 1203 1205 1814 1621 1627 Exp 1 Exp 2 1550 0.12 0.11 0.050.05 0.07 0.22 0.17 0.10 0.06 0.14 1551 0.41 0.67 0.04 0.05 0.11 0.370.33 0.11 0.07 0.15 1552 0.76 1.20 0.05 0.06 0.13 0.19 0.18 0.11 0.320.13 1553 1.07 1.24 0.65 0.65 0.85 0.17 0.17 0.14 0.41 0.15 1554 0.821.01 0.84 0.51 0.73 0.16 0.17 0.12 0.26 0.15 1555 0.11 0.12 0.24 0.260.28 0.26 0.32 0.29 0.30 0.45 1030* 1 1 1 1 1 1 1 1 1 1 *Full-lengthCD137

Example 13 In Vitro Efficacy of CD137 Antibodies

Material and Methods

Agonistic activity of CD137 antibodies was evaluated in a T cell assaybased on primary human CD8⁺ T cells. Briefly, CD8⁺ T cells wereseparated from human peripheral blood mononuclear cells by MACSseparation (Miltenyi #130-096-495) according to the manufacturer'sprotocol. Cells were incubated in 96-well microtiter plates (NuncThermoScientific #268200), pre-coated with anti-CD3 antibody (clone OKT3,Affymetrix eBioscience #16-0037) and titrated concentrations of theCD137 antibody to be tested. Following 72 or 96 hour incubation, culturemedium was harvested and IFN-γ levels were determined by ELISA (BD#555142).

Each clone was analyzed in at least 6 donors and compared to thereference CD137 antibody 1811/1812 and the negative control antibody.

Due to large intra-donor variations the stimulation index (SI, foldinduction by antibody compared to negative control) was determined foreach sample and normalized to the stimulation index for the referenceantibody 1811/1812.

Results and Conclusion

Several clones with efficacy comparable to the reference 1811/1812 wereidentified.

Data are summarized in FIG. 8, which indicates the absolute IFN-γ levelsinduced by CD137 stimulation. However, all antibodies were not analyzedhead-to-head in all donors, and the normalized SI is more relevant forcomparison of the efficacy. The antibodies were evaluated in an IgG1format, and the efficacy was measured using antibodies coated to thesurface of the wells, which may influence the efficacy.

Example 14 Competitive Binding of CD137 Antibodies (Ligand Blocking)

Aim and Background

The aim was to determine if the exemplary CD137 antibodies block theCD137 ligand binding.

In the previous domain mapping experiment, the CD137 antibodies weredivided in different groups based on their binding to similar subdomainsof the CD137 antigen. If the CD137 antibodies bind to epitopes close tothe ligand binding region, binding to the antigen can lead to partial ortotal blockade of ligand binding. Binding close to the CD137 ligandbinding epitope may also affect the ligand binding due to sterichindrance or conformational changes of the CD137 ligand binding epitope.All CD137 antibodies were titrated against a fixed concentration ofCD137L for evaluation of ligand blocking properties.

Material and Method

CHO-cells transfected with human CD137 were used for the ligandcompetition. The extracellular part of human CD137 was fused to thetransmembrane and intracellular part of hCD40 and cloned into pcDNA3.1.The vector was subsequently stably transfected into CHO cells. Theexpression of CD137 was confirmed by staining with commercial antibodytargeting CD137.

The CHO-huCD137 were pre-incubated with CD137 monoclonal antibodies,titrating down from 10:1 down to 0.01:1 molar ratio CD137 mAb (250μg/ml) to CD137L (hCD137_CD8 Ligand) (Ancell #503-020), for 1 h at +4Cbefore the addition of CD137 ligand at a concentration at EC50. Afterco-incubation for another 30 min at +4C, cells were washed and boundCD137 ligand was detected with αCD8a-PE (clone 53-6.7) (BD #553033) andfixed with paraformaldehyde (10× concentrate BD CellFIX, BDbiosciences). Analysis was performed with FACSverse and the MFI (MedianFluorescence Intensity) was calculated with FlowJo software

Results and Conclusion

The CD137L blocking experiment was performed in duplicate. It can beconcluded that not all CD137 mAbs tested were blocking the CD137 ligandbinding (Table 13, FIG. 9). CD137 mAbs belonging to group B and C (1204and 1620), binding to domain 2B-4A, were blocking the CD137L. Antibody1814 also blocks the CD137L binding. 1618, belonging to group A whichbound to domain 1, did not block CD137 ligand.

TABLE 13 Maximal CD137 ligand competition of the CD137 antibodies, meanout of two experiments Group CD137 CD137L, (domain mapping) mAb inhib. A1618  2% C 1814 67%

Example 15 Competitive Binding of CD137 Antibodies Measured by ELISA

Aim and Background

By competing the exemplary CD137 antibodies with each another, it ispossible to determine antibodies binding to similar epitopes based ontheir blocking pattern. The competition ELISA is performed byco-incubating biotinylated CD137 antibodies with non-biotinylated CD137antibodies when binding to coated CD137-Fc. Competition is defined asloss of signal from the biotinylated CD137 antibody. Low competitionvalues could either be due to no competition between the antibodies orbinding kinetics of the antibodies. Binding of one antibody could alsolead to steric hindrance or conformational changes when binding theantigen which affects the binding of the other CD137 antibody.

Material and Methods

CD137 antibodies were biotinylated (EZ-link NHS-LC-Biotin, ThermoFisher)and intact binding properties to CD137-Fc were verified with ELISA bycomparing EC50 between biotinylated and non-biotinylated anti-CD137mAbs. Non-biotinylated anti-CD137 (anti-CD137-bio) was pre-incubatedwith CD137-Fc at concentrations 30 times higher than the determined EC50for 0.5 h. Without washing, anti-CD137-bio was added and co-incubatedfor another 1 h. The binding of anti-CD137-bio was detected withStreptavidin-HRP (Pierce). Competition was calculated as the relativenumber by dividing the binding measured to other antibodies relative toits maximum competition (competing with itself). The relative valuesobtained were normalized against the maximum blocking capacity (Table4).

TABLE 14 Summary of CD137 antibody competition ELISA from twoexperiments. Values are presented as % competition with CD137-bio. Groupcomp Pattern X Pattern Y ELISA 1812 1618 1202 1204 1814 1620 1626 12141812-bio 100 100 7 5 5 4 0 4 1814-bio 15 21 41 74 94 61 57 99 1202-bio18 19 58 76 63 50 63 92 1214-bio 12 6 81 92 78 80 77 99 1618-bio 84 8811 3 6 10 16 9 1620-bio 4 7 49 93 100 82 79 100 1626-bio 37 24 100 10096 97 100 99 1204-bio 23 28 71 88 72 66 66 97

Result and Conclusion

When normalizing the relative competition values for each antibody acompetition pattern could be identified (Table 14). The antibodies 1812and 1618 displayed a unique pattern in the competition ELISA (PatternX). The other CD137 antibodies that were analyzed had a similar blockingpattern (Pattern Y). Differences in binding kinetics between thoseantibodies, may explain some of the minor variations in the bindingpatterns among these antibodies, although it cannot be excluded that thesmall variations within group Y reflects actual differences in thebinding epitope.

Example 16 Crosslinking dependency of CD137 mAbs Material and Methods

The crosslinking dependency of CD137 antibodies was evaluated in a Tcell assay based on primary human CD8⁺ T cells. Briefly, cells wereincubated in 96-well microtiter plates (NuncThermo Scientific #268200)pre-coated with anti-CD3 antibody (clone OKT3, Affymetrix eBioscience#16-0037). Titrated concentrations of CD137 antibodies in the presenceand absence of crosslinking antibody, goat-anti human Fc F(ab′)2(Jackson Immuno #109-006-098) at 1:3 molar ratio were added to theplated. Following 72, culture medium was harvested and IFN-γ levels weredetermined by ELISA (BD #555142).

Results and Conclusion

The results are summarised in FIG. 10 showing the CD137 activation inthe presence of crosslinking antibody and FIG. 11 showing the activationin the absence of crosslinking antibody. From the results obtained itcan be concluded that CD137 mAb clone 1618 is crosslinking dependent andthe reference, a CD137 specific IgG4 antibody (REF Ab), is crosslinkingindependent, when it comes to CD137 mediated activation ofCD137-expressing immune cells.

Example 17 Production of 5T4-CD137 Bispecific Antibodies

Materials and Methods

Thirty 5T4-CD137 bsAb, based on four 5T4 and eight CD137 antibodies werecloned as bsAb with one of the binding moieties cloned as a scFv andfused to the C-terminus of heavy chain of the IgG (i.e. in the Morrisonformat). The majority of the bsAb were clones with the 5T4 binder asscFv and the CD137 agonist as IgG, but in some constructs, a CD137 scFvwas fused to the heavy chain of a 5T4 IgG (Table 15). In addition, fourisotype control constructs, where either the 5T4 or the CD137 binder hadbeen replaced with an isotype control antibody were included. bsAb wereproduced by transient transfection of Freestyle293 cells (ThermoFischer) and purified by Protein A chromatography.

-   -   The bsAb designation was as follows:        -   First number indicates antibody clone name        -   Second number indicates scFv clone name

Thus, the designation “1200-1206” refers to the 5T4 binder 1206 (i.e.comprising the variable domain heavy and light chain sequences ofantibody 1206/1207) in scFv format fused to the C-terminus of the Fc ofthe CD137 agonist antibody 1200 (i.e. comprising the variable domainheavy and light chain sequences of antibody 1200/1201).

TABLE 15 List of all 5T4-CD137 bsAb that were cloned and produced forfurther evaluation mAb scFv Protein (clone (clone name mAb target name)scFv target name)  1 1200-1206 CD137 1200 5T4 1206  2 1200-1208 CD1371200 5T4 1208  3 1200-1210 CD137 1200 5T4 1210  4 1200-1212 CD137 12005T4 1212  5 1202-1206 CD137 1202 5T4 1206  6 1202-1208 CD137 1202 5T41208  7 1202-1210 CD137 1202 5T4 1210  8 1202-1212 CD137 1202 5T4 1212 9 1204-1206 CD137 1204 5T4 1206 10 1204-1208 CD137 1204 5T4 1208 111204-1210 CD137 1204 5T4 1210 12 1204-1212 CD137 1204 5T4 1212 131210-1202 5T4 1210 CD137 1202 14 1210-1204 5T4 1210 CD137 1204 151210-1214* 5T4 1210 CD137 1214 16 1212-1202 5T4 1212 CD137 1202 171212-1204 5T4 1212 CD137 1204 18 1212-1214 5T4 1212 CD137 1214 191206-1202 5T4 1206 CD137 1202 20 1206-1204 5T4 1206 CD137 1204 211208-1202 5T4 1208 CD137 1202 22 1208-1204 5T4 1208 CD137 1204 231214-1208 CD137 1214 5T4 1208 24 1618-1208 CD137 1618 5T4 1208 251620-1208 CD137 1620 5T4 1208 26 1626-1208 CD137 1626 5T4 1208 271214-1210 CD137 1214 5T4 1210 28 1618-1210 CD137 1618 5T4 1210 291620-1210 CD137 1620 5T4 1210 30 1626-1210 CD137 1626 5T4 1210 1862-1210Isotype control 1862 5T4 1210 1862-1212 Isotype control 1862 5T4 12121202-1862 CD137 1202 Isotype control 1862 1204-1862 CD137 1204 Isotypecontrol 1862 *No expression

Example 18 Binding to Human CD137 and 5T4 by 5T4-CD137 BispecificAntibodies Measured by ELISA

Materials and Methods

Bispecific binding to both targets, CD137 and 5T4, was evaluated using astandard ELISA protocol. Plates (#655074, Greiner Bio-One GmbH, Germany)were pre-coated with 0.5 μg/ml 5T4-Fc (obtained from Professor PeterStern, University of Manchester) overnight. CD137-5T4 bsAb were dilutedfrom 8 to 2×10⁻³ μg/ml in 1:4 dilutions and added in duplicates of 50 μlto each well. CD137-bio (Ancell #502-030) was used as detection antibodyat 0.5 μg/ml and the binding was detected with Streptavidin-HRP (Pierce#21126). The ELISA was developed with SuperSignal ELISA PICOChemiluminescent substrate (Thermo Scientific Pierce, Rockford, Ill.USA) during 2-10 minutes and read in an automated microplate basedmulti-detection reader (FLUOstar OPTIMA, Netherlands).

Results and Conclusions

The majority of the bsAb bound to both targets in dual ELISA with EC50values at sub- or low nM range. However, some bsAb exhibitedconsiderably higher EC50 values, indicating poor affinity to either orboth targets in this antibody—scFv combination. Dose response curves areshown in FIG. 12 and EC50 values are summarized in Table 16.

TABLE 16 EC50 values of dual binding of 5T4-CD137 bispecific antibodies95% Confidence EC50 Intervals Clone (nM) (nM) 1200-1206 2.17 1.3-3.71202-1206 0.61 0.3-1.2 1204-1206 0.54 0.3-0.9 1206-1202 0.28 0.2-0.41206-1204 0.66 0.4-1.1 1200-1208 16.82  7.9-35.8 1202-1208 2.25 1.1-4.71204-1208 2.34 1.2-4.6 1214-1208 1.40 0.5-4.1 1618-1208 1.47 0.4-5.21620-1208 12.70  5.7-28.4 1626-1208 1.91 0.6-5.9 1208-1202 1.32 0.7-2.51208-1204 1.21 0.7-2.1 1200-1210 3.73 2.6-5.3 1202-1210 1.20 0.6-2.41204-1210 0.73 0.4-1.4 1214-1210 0.19 0.1-0.5 1618-1210 0.20 0.1-0.61620-1210 1.74 0.7-4.1 1626-1210 0.40 0.1-1.2 1210-1202 0.28 0.1-0.61210-1204 0.28 0.1-0.5 1200-1212 1.55 1.0-2.3 1202-1212 1.67 1.0-2.71204-1212 1.01 0.6-1.6 1212-1202 0.57 0.4-0.9 1212-1204 0.27 0.2-0.51212-1214 0.79 0.5-1.3

Example 19 Affinity of CD137-5T4 Bispecific Antibodies Measured bySurface Plasmon Resonance

Materials and Methods

Binding kinetics of a selection of the CD137-5T4 bsAbs was evaluatedusing the SPR-based MASS-1 platform (Sierra Sensors). Briefly, CD137 or5T4 was captured at the sensor chip surface using a streptavidin coatedchip and biotinylated antigen. The different CD137-5T4 bsAbs were theninjected over the chip in increasing concentrations and the associationand dissociation rates studied in real time.

Results and Conclusions

A summary of the obtained binding rate constants and affinities obtainedis presented in Table 17. It should be taken into consideration that theassay setup used allows for bivalent binding of the bsAbs to theantigen. This will give rise to avidity effects that lead to asignificant underestimation of the off-rates (kd) and thus also theaffinity value (KD). This makes comparisons to other compoundstroublesome, but the obtained values are valid for comparisons withinthe dataset.

The results from the kinetics analysis confirm retained affinity of theCD137-specific mAb part of the bispecific molecule, while the scFv partdisplays reduced 5T4 affinity as compared to the parental mAb. Asexpected, the conformational changes induced by a flexibility reducinglinker in the scFv format has a negative effect on the antigen bindingaffinity. In the case of 1210 this effect is only minor, while theaffinity of 1208 is reduced about 6 times.

TABLE 17 Summary of binding kinetics of CD137/5T4-specific bsAbsparental mAb KD bsAb Antigen Fit model ka (1/Ms) kd (1/s) KD (M) (M)1618- CD137 1:1 9.40E+ 1.09E− 1.16E− 1.56E− 1208 Langmuir 05 04 10 101618- 5T4 1:1 7.65E+ 4.62E− 6.01E− 9.63E− 1208 Langmuir 04 05 10 111618- CD137 1:1 1.26E+ 1.62E− 1.29E− 1.56E− 1210 Langmuir 06 04 10 101618- 5T4 1:1 4.86E+ 4.55E− 9.37E− 4.37E− 1210 Langmuir 05 04 10 10

Example 20 Functional Activity of 5T4-CD137 Bispecific Antibodies onHuman CD8+ T cells cultured in 5T4-Fc coated plates

Materials and Methods

The functional activity of 5T4-CD137 bsAb was evaluated in a CD8 T cellassay, where cells were cultured in microtiter plates coated with 5T4-Fcand CD3 antibody. Peripheral blood mononuclear cells (MNC) were isolatedby density gradient centrifugation using Ficoll-Paque (p 1.077 g/m1) (GEHealthcare #17-1440-02) from leucocyte concentrates obtained fromhealthy donors (Clinical Immunology and Transfusion Medicine, LabmedicinRegion Sickle, Lund Sweden). CD8⁺ T cells were enriched by negativeselection using the CD8⁺ T cell isolation kit (Miltenyi 130-096-495).Plates were coated overnight at 4° C. with 3 μg/ml αCD3, clone OKT3(Affymetrix eBioscience #16-0037-85), washed and coated with 5 μg/ml5T4-Fc for 2 h at 37° C. After the 5T4-Fc coating, plates were washedand blocked for a minimum of 30 minutes with RPMI (Gibco #61870010)containing 10% FCS (Heat inactivated, Gibco #10270-106 lot 41Q9248K) and10 mM Hepes (Gibco #15630056).

CD137-5T4 bsAbs were diluted in RPM! containing 10% FCS and 10 mM Hepesand added to the plates 30 minutes before addition of CD8⁺ T cells(0.07×10⁶ cells/well). Assay plates were incubated for either 68 or 92 hat 37° C. and culture supernatant harvested. IFN-γ levels in thesupernatants were measured by ELISA (BD OptiEIA #555142). Results areshown as fold change compared to CE_1200-1210, which was used as aninternal control in all experiments.

Results and Conclusions

Results from the first set of bsAbs used at a fixed concentration of 1μg/ml (FIG. 13) show that the majority of the bsAbs based on either ofthe 5T4 binders 1206, 1208 or 1210 were functional in the T cell assay,whereas those that were based on the 5T4 antibody 1212 were not. Dataalso suggest that bsAb based on CD137 clone 1202 may have lower efficacyand/or potency compared to bsAb based on CD137 clones 1204 and 1200. Theagonistic effect of the 5T4-CD137 bsAb was dependent on cross-linking by5T4, since no activation was obtained in the absence of 5T4 or usingbsAb comprising one isotype control moiety.

Based on these results, a second set of bsAbs based on five new CD137clones as IgG and the 5T4 clones 1208 and 1210 as scFv wereinvestigated. 5T4 binders 1212 and 1206 were excluded due to poorfunctional activity as bsAb, and low Tm value and thus poor stability asscFv, respectively. Functional activity of all bsAb is summarized inFIG. 14.

Example 21 Functional Activity of 5T4-CD137 Bispecific Antibodies onHuman CD8+ T cells Cultured with 5T4-Expressing Tumor Cells

Materials and Methods

CD8 T cells were isolated as described above, and cultured in thepresence of 5T4-expressing cells B16 cells. B16 cells transfected withempty vector were used as negative control. CD3 stimulation wasperformed with αCD3 (OKT-3) coated beads (Dynal M-450 Tosylactivated#14013) according to the manufactures protocol.

Irradiated B16 tumor cells (6000 cells/well) were added to the 96 wellplates and let to attached for 2 h. CD137-5T4 bsAbs were added andincubated for 30 minutes prior to addition of CD8⁺ T cells (0.1×10⁶cells/well) and αCD3 coated beads (0.5×10⁵ beads/well). Plates werecultured for 68 or 92 h and IFN-γ levels in the media measured by ELISA(BD OptiEIA #555142).

Results and Conclusions

Results from the fully cell-based assay show that the majority of thebsAb are functional and that the effect is 5T4-specific, with noactivation induced by 5T4 negative B 16 cells or isotype-CD137 bsAb(FIG. 15).

Example 22 Optimization of Affinity and Biophysical Properties ofBispecific Antibodies: Optimization of 5T4-Specific Variable Domains

Material and Methods

The aim of the optimization was to generate versions of the 5T4-specific1210/1211 antibody in regards to affinity and biophysical properties.Selections were performed towards 5T4 with lead optimized library1210LOlib1. In total, 170 unique clones were identified in the initialprimary screening with good target signal as well as target/non-targetratio. These clones were further investigated in an extended primaryscreening with regards to temperature stability. The temperaturestability evaluation showed that the majority of the identified uniqueclones displayed a better stability compared to the wild type 1210/1211scFv clone. The top 96 clones were further evaluated in a dose-responseELISA and they all showed a similar and acceptable binding behaviour.Sequence analysis from the test-screening and primary screening showedsimilar trends.

The top 96 identified clones from the primary and the extended primaryscreening were further re-cloned as the scFv-part in theMorrison-format, with 1618/1619 as the monoclonal antibody (mAb) part,and were evaluated based on binding, affinity and stability.

Kinetic measurements were performed using the Octet RED96 platformequipped with Anti-human Fab-CH1 2^(nd) generation sensor tips(ForteBio). Bispecific antibodies were diluted to 1.5 μg/ml in 1×kinetic buffer (ForteBio) and coupled to the biosensors. Human 5T4(produced in-house) was diluted in 1× Kinetics Buffer to 50 nM, 10 nMand 2 nM. Binding kinetics were studied in 1× Kinetics buffer whereassociation was allowed for 300 sec followed by dissociation for 600sec. Sensor tips were regenerated using 10 mM glycine, pH 2.2. Datagenerated were referenced by subtracting a parallel buffer blank, thebaseline was aligned with the y-axis, inter-step correlation byalignment against dissociation was performed and the data were smoothedby a Savitzky-Golay filter in the data analysis software (v.9.0.0.14).The processed data were fitted using a 1:1 Langmuir binding model withX² as a measurement of fitting accuracy.

Results and Conclusions

The data are summarised in Table 18. Overall, the lead optimizedvariants behaved very similarly in the EC50 evaluation in ELISA. Theaffinity evaluation showed that the affinity (KD) had been improvedbetween 3 to more than 10 times compared to the wild type 1618-1210clone. The EC50 evaluation on cells also showed similar behaviour of thedifferent optimized variants and all showed an improved performancecompared to the wild type 1618-1210 clone. In regards to stability, thetop performing clones show less than 10% aggregation after protein Apurification and have a Tm higher than the Tm of the Fc (>70° C.) asmeasured by HPLC and DSF respectively.

TABLE 18 Summary of affinity measurements of optimized 5T4-specificvariable domains Composition of construct (amino acid sequences) A B C DAffinity (VH of (VL of (VH (VL of Additional measurements Octet (5T4)Antibody name B1) B1) of B2) B2) Connector* alteration* KD (M) kon(1/Ms)kdis(1/s) 1618-1210LO1 1618 1619 2992 2993 m6 m2 1.47E−10 2.23E+053.29E−05 1618-1210LO2 1618 1619 2994 2995 m6 m2 2.70E−10 1.67E+054.50E−05 1618-1210LO3 1618 1619 2996 2997 m6 m2 3.49E−10 2.12E+057.40E−05 1618-1210LO4 1618 1619 2998 2999 m6 m2 3.82E−10 2.02E+057.73E−05 1618-1210LO5 1618 1619 3000 3001 m6 m2 4.67E−10 1.64E+057.65E−05 1618-1210LO6 1618 1619 3002 3003 m6 m2 4.69E−10 2.27E+051.06E−04 1618-1210LO7 1618 1619 3004 3005 m6 m2 4.95E−10 1.68E+058.29E−05 1618-1210LO8 1618 1619 3006 3007 m6 m2 4.98E−10 1.77E+058.83E−05 1618-1210LO9 1618 1619 3008 3009 m6 m2 5.34E−10 2.35E+051.25E−04 *see Table D(5) and D(6) for details

Example 23 Optimization of Affinity and Biophysical Properties ofBispecific Antibodies: Optimization of CD137-Specific Variable Domains

Material and Methods

The aim of the optimization was to generate versions of theCD137-specific 1618/1619 antibody in regards to affinity and biophysicalproperties. Selections were performed towards CD137 with lead optimizedlibrary 1618LOlib1. In total, 153 unique clones were identified in theinitial primary screening with good target signal as well astarget/non-target ratio. These clones were further investigated in anextended primary screening with regards to temperature stability. Thetemperature stability evaluation allowed for identification of the bestperforming unique clones in regards to temperature stability compared tothe wild type 1618/1619 scFv clone. The top 50 clones were furtherevaluated in a dose-response ELISA and they all showed a similar andacceptable binding behaviour. Sequence analysis from the test-screeningand primary screening showed similar trends.

The top 50 identified clones from the primary and the extended primaryscreening were further re-cloned as the scFv-part in theMorrison-format, with 1210/1211 as the monoclonal antibody (mAb) part,and were evaluated based on binding, affinity and stability. Kineticmeasurements were performed using the Octet RED96 platform equipped withAnti-human Fab-CH1 2^(nd) generation sensor tips (ForteBio). Bispecificantibodies were diluted to 1.5 μg/ml in 1× kinetic buffer (ForteBio) andcoupled to the biosensors. Human CD137-Fc (R&D Systems, #838-4B) wasdiluted in 1× Kinetics Buffer to 50 nM, 10 nM and 2 nM. Binding kineticswere studied in 1× Kinetics buffer where association was allowed for 300sec followed by dissociation for 600 sec. Sensor tips were regeneratedusing 10 mM glycine, pH 2.2. Data generated were referenced bysubtracting a parallel buffer blank, the baseline was aligned with they-axis, inter-step correlation by alignment against dissociation wasperformed and the data were smoothed by a Savitzky-Golay filter in thedata analysis software (v.9.0.0.14). The processed data were fittedusing a 1:1 Langmuir binding model with X² as a measurement of fittingaccuracy.

Results and Conclusions

The data are summarised in Table 19. Overall, the lead optimizedvariants behaved very similarly in the EC50 evaluation in ELISA. Theaffinity evaluation showed that the affinity (KD) were comparable to thewild type 1210-1618 clone. The EC50 evaluation on cells also showedsimilar behaviour of the different optimized variants. In regards tostability, the top performing clones show less than 6% aggregation afterprotein A purification and have a Tm between 54° C.-59° C. as measuredby HPLC and DSF respectively.

TABLE 19 Summary of affinity measurements of optimized CD137-specificvariable domains Composition of construct (amino acid sequences) A B C DAffinity measurements Octet (VH of (VL of (VH of (VL of Additional(CD137) Antibody name B1) B1) B2) B2) Connector* alteration* KD (M)kon(1/Ms) kdis(1/s) 1210-1618LO1 1210 1211 3012 3013 m6 m2 1.58E−092.18E+05 3.44E−04 1210-1618LO2 1210 1211 3014 3015 m6 m2 1.64E−092.81E+05 4.61E−04 1210-1618LO3 1210 1211 3016 3017 m6 m2 1.90E−093.45E+05 6.54E−04 1210-1618LO4 1210 1211 3018 3019 m6 m2 2.38E−092.89E+05 6.87E−04 1210-1618LO5 1210 1211 3020 3021 m6 m2 2.56E−092.62E+05 6.72E−04 1210-1618LO6 1210 1211 3022 3023 m6 m2 2.57E−092.99E+05 7.68E−04 1210-1618LO7 1210 1211 3024 3025 m6 m2 2.86E−092.85E+05 8.14E−04 1210-1618LO8 1210 1211 3026 3027 m6 m2 2.98E−092.19E+05 6.53E−04 1210-1618LO9 1210 1211 3028 3029 m6 m2 3.24E−093.32E+05 1.08E−03 1210-1618LO10 1210 1211 3030 3031 m6 m2 3.38E−092.80E+05 9.48E−04 1210-1618LO11 1210 1211 3032 3033 m6 m2 3.66E−092.89E+05 1.06E−03 1210-1618LO12 1210 1211 3034 3035 m6 m2 2.38E−093.83E+05 9.11E−04 1210-1618LO13 1210 1211 3036 3037 m6 m2 2.82E−093.52E−05 9.95E−04 *see Table D(5) and D(6) for details

Example 24 Optimization of Affinity and Biophysical Properties ofBispecific Antibodies: Dual ELISA Analysis of Optimized BispecificAntibodies

Material and Methods

Optimized bispecific antibodies with improved biophysical propertieswere obtained using different strategies including combining leadoptimized binding domains and the use of additional mutations andconnectors.

The bispecific antibody in this example is an IgG-scFv bispecificantibody. The CD137 binding domain is an intact IgG and the 5T4 bindingdomain is an scFv attached to the C-terminus of a heavy chain of theIgG. The bispecific antibodies comprise for example the followingcomponents: (1) Two heavy chains each comprising, in order from theN-terminus to the C terminus: [a VH sequence; A in Table 20]-[an H chainconstant region of IgG1 subtype with no mutations unless stated by an mXsuffix in Table 20]-[an m6, m15, m16 or m17 connector ]-[a scFv, whereinthe variable chains (heavy or light) are ordered from the N-terminus tothe C terminus so that chain C in Table 20 is followed by a linker andthen followed by Chain D in Table 20]; and (2) Two light chains eachcomprising, in order from the N-terminus to the C terminus: [a VLsequence; B in Table 20]-[an L chain constant region].

The scFv for some of the bispecific antibodies in this example carryrecombinant N-glycosylation sites placed either in the

Optimized bispecific antibody encoding genes were designed in house andsynthesized at GeneArt (Thermo Fisher, Life Technologies) or generatedby standard cloning methods into expression vectors. Bispecificantibodies were produced by transient transfection of Expi293™ (ThermoFischer Scientific) and purified by Protein A chromatography. Bispecificbinding to both targets, CD137 and 5T4, was evaluated using a standardELISA protocol. Plates (#655074, Greiner Bio-One GmbH, Germany) werepre-coated with 0.5 μg/ml 5T4-Fc (obtained from Professor Peter Stern,University of Manchester) overnight. CD137-5T4 bsAb were diluted from 8to 2×10⁻³ μg/ml in 1:4 dilutions and added in duplicates of 50 μl toeach well. CD137-bio (Ancell #502-030) was used as detection antibody at0.5 μg/ml and the binding was detected with Streptavidin-HRP (Pierce#21126). The ELISA was developed with SuperSignal ELISA PICOChemiluminescent substrate (Thermo Scientific Pierce, Rockford, Ill.USA) during 2-10 minutes and read in an automated microplate basedmulti-detection reader (FLUOstar OPTIMA, Netherlands).

Results and Conclusions

The data are summarised in Table 20. The optimized bispecificantibodies, consisting of lead optimized CD137 binding domains and/orlead optimized 5T4 binding domains and/or stabilised bispecificantibodies using novel connectors and/or additional stabilisingstrategies including reversed heavy and light chain order orN-glycosylation sites, display dual binding for both targets, CD137 and5T4. Binding domains with improved binding such as for example 1210L01and 1210L02 provide improved dual binding as observed as lower EC50values compared to bispecific antibodies comprising non-optimized 1210binding domains.

TABLE 20 Summary of Dual ELISA measurements of optimizedCD137/5T4-specific bsAbs Composition of construct (amino acid sequence)C A B (VH D (VH of (VL of of (VL of Additional EC50 Dual Antibody nameB1) B1) B2) B2) Connector* alteration* ELISA 1618LO1-1210LO1 3012 30132992 2993 m6 m2 0.5 1618LO1-1210LO2 3012 3013 2994 2995 m6 m2 0.51618LO3-1210LO1 3016 3017 2992 2993 m6 m2 0.5 1618LO3-1210LO2 3016 30172994 2995 m6 m2 0.4 1618LO3-1210 3016 3017 1210 1211 m6 m2 0.91618LO11-1210LO1 3032 3033 2992 2993 m6 m2 0.5 1618LO11-1210LO2 30323033 2994 2995 m6 m2 0.6 1618LO11-1210 3032 3033 1210 1211 m6 m2 1.11210-1618.m2.m15 1210 1211 1618 1619  m15 m2 0.4 1210-1618.m2.m16 12101211 1618 1619  m16 m2 0.4 1210-1618.m2.m7.m15 1210 1211 1619 1618  m15m2 0.7 1210-1618.m2.m7.m17 1210 1211 1619 1618  m17 m2 0.61210-1618.m2.m7.m18 1210 1211 1619 1618  m18 m2 0.4 1618-1210.m2.m151618 1619 1210 1211  m15 m2 0.5 1618-1210.m2.m16 1618 1619 1210 1211 m16 m2 0.5 1618-1210.m2.m17 1618 1619 1210 1211  m17 m2 0.51618-1210.m2.m7.m15 1618 1619 1211 1210  m15 m2 5.3 1618-1210.m2.m7.m161618 1619 1211 1210  m16 m2 3.7 1618-1210.m2.m7.m17 1618 1619 1211 1210 m17 m2 1.2 1618-1210.m2.m7.m18 1618 1619 1211 1210  m18 m2 2.61618-1210.m2.m6.m19 1618 1619 1210 1211 m6 m2, m19 0.41618-1210.m2.m6.m20 1618 1619 1210 1211 m6 m2, m20 0.4 *see Table D(5)and D(6) for details

Example 25 Binding of Lead Optimised 5T4 Clones to Cells Expressing 5T4,Measured by Flow Cytometry

Materials and Methods

Analysis of 5T4 mAb binding with flow cytometry was performed usinghuman and Macaca mulatta (cynomolgus) 5T4-transfected CHO-K cell linesand as negative control, mock transfected cells. Cells were stained with5T4 lead optimised clones (scFv in bsAb format) diluted in FACS buffer(PBS, 0.5% BSA and 0.02% NaN₃). Binding was detected with the secondaryantibody anti-IgG (Fc)-PE (109-115-098, Jackson ImmunoResearch Europe,UK) diluted 1:100. Samples were run on a FACSverse (BD Biosciences,Heidelberg, Germany) and mean fluorescence intensity (MFI) wasdetermined using the FlowJo software.

ELISA was performed using a standard protocol. Plates (#655074, GreinerBio-One GmbH, Germany) were pre-coated with 0.5 μg/ml 5T4-Fc (producedin-house) overnight. 5T4 antibodies were diluted in PBST+1% BSA and 50μl was added to each well. Binding was detected with anti-human kappalight chain antibody (AbD Serotec #STAR127P) and the ELISA was developedwith SuperSignal ELISA PICO Chemiluminescent substrate (ThermoScientific Pierce, Rockford, Ill. USA) for 2-10 minutes and read in anautomated microplate based multi-detection reader (FLUOstar OPTIMA,Netherlands).

Results and Conclusions

Binding curves for CHOh5T4 and CHOcyno5T4 cells can be seen in FIG. 16(A and B). All the lead optimized variants have similar binding potencytowards both human and cyno 5T4 expressing cells as well as towardshuman 5T4 measured with ELISA compared to the original antibody. Thelead optimized variants have an improved affinity for both human andcyno 5T4 as measured with both ELISA and FACS.

Example 26 Binding of Lead Optimised CD137 Clones to Cells ExpressingCD137, Measured by Flow Cytometry

Material and Methods

Analysis of CD137 mAb binding with flow cytometry was performed usinghuman and cyno CD137-transfected CHO-K cell lines and as negativecontrol, mock transfected cells. Cells were stained with CD137 leadoptimised clones (as scFv in bsAb format) diluted in FACS buffer (PBS,0.5% BSA and 0.02% NaN₃). Binding was detected with the secondaryantibody anti-IgG (Fc)-PE (109-115-098, Jackson ImmunoResearch Europe,UK) diluted 1:100. Samples were run on a FACSverse (BD Biosciences,Heidelberg, Germany) and mean fluorescence intensity (MFI) wasdetermined using the FlowJo software.

ELISA plates (Greiner #655074) were coated with 50 μl/well ofrecombinant CD137 (R&D #838-4B) diluted to a final concentration of 0.5μg/ml in PBS for 1 h at 37° C. or overnight at 4° C. Plates were washedwith PBS+0.05% TWEEN20 (PBST), followed by block with PBST+1% bovineserum albumin (BSA). Antibody samples were diluted in PBST+1% BSA andincubated for 1 h in room temperature, followed by detection using ahorse radish peroxidase-conjugated anti-human kappa light chain antibody(AbD Serotec #STAR127P) and developed using SuperSignal ELISA PicoChemiluminescent substrate (Pierce ThermoScientific #37069).

Results and Conclusions

Binding curves of the bispecific antibodies to CHOhCD137 andCHOcynoCD137 cells can be seen in FIG. 17 (A and B).

The EC50 values are comparable to the wild type 1210-1618 (1618 as scFv)clone for both human and cyno CD137.

Example 27 In Vitro Activity of Lead Optimised 5T4-CD137 (1618-1210)Bispecific Antibodies in an IFNγ Release Assay Using Human CD8+ T cellson 5T4 coated plates

Material and Methods

The functional activity of the 5T4-CD137 bsAb was evaluated in a CD8+ Tcell assay, where cells were cultured in microtiter plates coated with5T4-Fc and CD3 antibody. Peripheral blood mononuclear cells (MNC) wereisolated by density gradient centrifugation using Ficoll-Paque (p1.077g/ml) (GE Healthcare #17-1440-02) from leucocyte concentratesobtained from healthy donors (Clinical Immunology and TransfusionMedicine, Labmedicin Region Skåne, Lund Sweden). CD8+T cells wereenriched by negative selection using the CD8+T cell isolation kit(Miltenyi 130-096-495). Plates were coated overnight at 4° C. with 3μg/ml αCD3, clone OKT3 (Affymetrix eBioscience #16-0037-85), washed andcoated with 5 μg/ml 5T4-Fc for 2 h at 37° C. After the 5T4-Fc coating,plates were washed and blocked for a minimum of 30 minutes with RPMI(Gibco #61870010) containing 10% FCS (Heat inactivated, Gibco #10270-106lot 41Q9248K) and 10 mM Hepes (Gibco #15630056). 1618-1210 bsAb wasdiluted in RPMI containing 10% FCS and 10 mM Hepes and added to theplates 30 minutes before addition of CD8+ T cells (0.07×10⁶ cells/well).Assay plates were incubated for 68 h at 37° C., and culture supernatantharvested. IFN-γ levels in the supernatants were measured by ELISA (BDOptiEIA #555142).

Results and Conclusions

The potency of the 1618-1210 bsAb was determined to EC50 0.6-0.9 nMusing human CD8+ T cells cultured in 5T4-Fc coated plates and was basedon two experiments and a total of six donors. The data were normalisedand the EC50 was determined using a three-parameter sigmoidaldose-response model (FIG. 18).

Bispecific antibodies variants with optimized variable domains of1618-1619 and 1210-1211 were generated as outlined in Table D, Table E,Table 18, Table 19 and Table 20. The generated bsAbs variants withoptimized variable sequences were functional in the CD8+ T cell assaywith crosslinked 5T4-Fc as seen in FIG. 19. To correlate the resultsfrom the different assay plates the calculated IFNγ levels werenormalised to a plate reference.

Bispecific antibodies were also generated with different linkers asoutlined in the Table D, Table E and Table 20, and were evaluated in theCD8 T cell assay. As shown in FIG. 20 the generated bsAbs could induce aCD137 activation only in the presence of the tumour antigen. As inprevious FIG. 19, the obtained IFNγ values were normalised to a positivecontrol in the plate.

Example 28 In Vitro Activity of Lead Optimised 5T4-CD137 (1618-1210)Bispecific Antibodies in an IFNγ Release Assay Using Human CD8+T CellsCultured with 5T4-expressing tumor cells (B16-17)

Material and Methods

CD8+ T cells were isolated as described above, and cultured in thepresence of tumour associated antigen (TAA) 5T4-expressing cells B16cells. B16 cells transfected with empty vector were used as negativecontrol. CD3 stimulation was performed with αCD3 (OKT3) coated beads(Dynal M-450 Tosylactivated #14013) according to the manufacturer'sprotocol.

Irradiated B16 tumour cells (6000 cells/well) were added to the 96 wellplates and left to attach for 2 hours. CD137-5T4 bsAbs were added andincubated for 30 minutes prior to addition of CD8+T cells (0.1×10⁶cells/well) and αCD3 coated beads (0.5×10⁵ beads/well). Plates werecultured for 68 hours and IFN-γ levels in the media were measured byELISA (BD OptiEIA #555142).

Results and Conclusions

The bispecific antibody 1618-1210 induced IFNγ production in CD8+ Tcells in a dose dependent manner when cultured on cells expressing 5T4(TAA), but not when cultured on cells that do not express 5T4. Theresults further confirm that CD137-TAA antibodies stimulate T cells onlyin the presence of tumour antigens. The potency of 1618-1210 bsAb wasdetermined to EC50 0.2-0.7 nM in the CD8+ T cell assay performed withB16-5T4 expressing tumour cells. The EC50 was based on the normaliseddata from two donors and determined by using a three-parameter sigmoidaldose-response model (FIG. 21).

Example 29 In Vitro Activity of Lead Optimised 5T4-CD137 (1618-1210)Bispecific Antibodies in an IFNγ Release Assay Using Human PBMCsCultured in 5T4-Fc Coated Plates

Material and Methods

The functional activity of 5T4-CD137 bsAb was evaluated in a PBMC assay,where cells were cultured in microtiter plates coated with 5T4-Fcantibody. Peripheral blood mononuclear cells (PBMC) were isolated bydensity gradient centrifugation using Ficoll-Paque (p 1.077g/ml) (GEHealthcare #17-1440-02) from leucocyte concentrates obtained fromhealthy donors (Clinical Immunology and Transfusion Medicine, LabmedicinRegion Skåne, Lund Sweden). CD8+ T cells were enriched by negativeselection using the CD8+ T cell isolation kit (Miltenyi 130-096-495).Plates were coated with 5 μg/ml 5T4-Fc for 2 h at 37° C. After the5T4-Fc coating, plates were washed and blocked for a minimum of 30minutes with RPMI (Gibco #61870010) containing 10% FCS (Heatinactivated, Gibco #10270-106 lot 41Q9248K) and 10 mM Hepes (Gibco#15630056).

1618-1210 bsAb were diluted in RPMI containing 10% FCS and 10 mM Hepesand added to the plates 30 minutes before addition of CD8+ T cells(0.1×10⁶ cells/well). CD3 stimulation was performed with 1 μg/ml solubleαCD3. Assay plates were incubated for 68 hours at 37° C., and culturesupernatant harvested. IFN-γ levels in the supernatants were measured byELISA (BD OptiEIA #555142).

Results and Conclusions

The results shown in FIG. 22 demonstrate that the bispecific antibody1618-1210.m2 induced a TAA (5T4)-dependent CD137 mediated activation ofPBMCs. No activation of PBMCs was detected without 5T4 present in theassay.

Example 30 In Vitro Activity of Lead Optimised 5T4-CD137 BispecificAntibodies in an IFNγ Release Assay Using Human CD8+ T Cells Culturedwith CD32 (FcγRII)-expressing L cells

Material and Methods

CD8 T cells were isolated as described above, and cultured in thepresence of CD32-expressing L cells. CD3 stimulation was performed withαCD3 (OKT3) coated beads (Dynal M-450 Tosylactivated #14013) accordingto the manufacturer's protocol.

Irradiated CD32 L cells (10000 cells/well) were added to the 96 wellplates and left to attach for 2 hours. CD137 (1618) mAb with and withoutthe LALA mutation was added and incubated for 30 minutes prior toaddition of CD8+ T cells (0.1×10⁶ cells/well) and αCD3 coated beads(0.5×10⁵ beads/well). Plates were cultured for 68 hours and IFN-γ levelsin the media were measured by ELISA (BD OptiEIA #555142).

Results and Conclusions

Results from the co-culture assay of CD32-expressing cells with CD8+ Tcells, shown in FIG. 23, demonstrate that CD137 activation is onlyinduced by 1618 containing the wt IgG1 and not by the Fc silenced 1618IgG1 containing the LALA mutation, further supporting the conclusionthat activation of T cells via CD137 with antibodies such as 1618/1619requires cross linking.

Example 31 Binding of TAA-CD137 Bispecific Antibodies Measured byDual-Binding ELISA

Material and Methods

Bispecific antibodies against three tumor associated antigens (TAA),EpCAM, HER2 and EGFR were generated. A scFv (1204/1205) binding to CD137was fused to the C-terminal end of three different IgG antibodies withthe sequences corresponding to the binding domains of Edrecolomab,Cetuximab and Herceptin. bsAbs were produced by transient transfectionof Expi293™ (Thermo Fischer Scientific) and purified by Protein Achromatography.

Bispecific binding to both targets, CD137 and TAA, was evaluated using astandard ELISA protocol. Plates (#655074, Greiner Bio-One GmbH, Germany)were pre-coated with 0.5 μg/ml TAA (hEGFR-His,SinoBiological#10001-H08H, hEpCAM-Fc, SinoBiological #10694-H02H,HER2-His, SinoBiological #10004-H08H and 5T4-Fc) overnight. TAA-bsAbwere diluted from 20 μg/ml in 1:4 dilutions and added in duplicates of50 μl to each well. CD137-bio (Ancell #502-030) was used as detectionantibody at 0.5 μg/ml and the binding was detected with Streptavidin-HRP(Pierce #21126). The ELISA was developed with SuperSignal ELISA PICOChemiluminescent substrate (Thermo Scientific Pierce, Rockford, Ill.USA) during 2-10 minutes and read in an automated microplate basedmulti-detection reader (FLUOstar OPTIMA, Netherlands).

Results and Conclusions

The generated TAA-CD137 bsAbs bound to both targets in the dual ELISAwith EC50 values in the low nM range (Table 21, FIG. 24).

TABLE 21 Summary of the generated TAA-CD137 bsAbs mAb scFv bsAb nameEpCam (2414) CD137 (1204) 2414-1204 EGFR (2424) CD137 (1204) 2424-1204Her2 (2078) CD137 (1204) 2078-1204

Example 32 In Vitro Activity of TAA-CD137 Bispecific Antibodies in anIFNγ Release Assay Using Human CD8+T Cells Cultured in TAA Coated Plates

Material and Methods

The functional activity of the three TAA-CD137 bsAbs binding to EpCAM,EGFR and Her2 was evaluated in a CD8+ T cell assay, where cells werecultured in microtiter plates coated with CD3 antibody and either EGFR,EpCam or Her2. As negative controls, parallel wells were coated withonly CD3 antibody. Peripheral blood mononuclear cells (PBMCs) wereisolated by density gradient centrifugation using Ficoll-Paque (p 1.077g/ml) (GE Healthcare #17-1440-02) from leucocyte concentrates obtainedfrom healthy donors (Clinical Immunology and Transfusion Medicine,Labmedicin Region Skåne, Lund Sweden). CD8+ T cells were enriched bynegative selection using the CD8+ T cell isolation kit (Miltenyi130-096-495). Plates were coated overnight at 4° C. with 3 μg/ml αCD3,clone OKT3 (Affymetrix eBioscience #16-0037-85), washed and coated with5 μg/ml TAA for 2 h at 37° C. After the TAA coating, plates were washedand blocked for a minimum of 30 minutes with RPMI (Gibco #61870010)containing 10% FCS (Heat inactivated, Gibco #10270-106 lot 41Q9248K) and10 mM Hepes (Gibco #15630056).

TAA-CD137 bsAbs were diluted in RPMI containing 10% FCS and 10 mM Hepesand added to the plates 30 minutes before addition of CD8+ T cells(0.07×10⁶ cells/well). Assay plates were incubated for 68 hours at 37°C., and culture supernatant harvested. IFN-γ levels in the supernatantswere measured by ELISA (BD OptiEIA #555142).

Results and Conclusions

The functionality of the EpCAM-1204 (FIG. 25(A)), EGFR-1204 (FIG. 25(B))and Her2-CD137 (FIG. 25(C)) bsAbs was analysed and it was concluded thatall of the generated bsAbs induced TAA mediated CD137 activation in thepresence of TAA and not in the absence of TAA (wells coated with onlyCD3 antibody). This strongly indicates that the TAA-dependentCD137-mediated immune cell activation generated by CD137-TAA antibodiesis a general phenomenon applicable to all types of cell surfaceexpressed TAA.

Example 33 In Vivo Anti-Tumor Effect of Bispecific Antibody 1618-1210 ina CT26-5T4 Colon Cancer Model

Summary

The anti-tumor effect of 1618-1210 (an exemplary antibody targetingCD137 and 5T4) was investigated using transgenic mice for human CD137and subcutaneous syngeneic tumor model of CT26 colon carcinomaexpressing human 5T4.

The bispecific antibody 1618-1210 demonstrated tumor volume inhibitioncompared to monoclonal antibody 1618 targeting CD137.

Material and Methods

Human 4-1BB knock-in mouse model was developed by Prof. Lieping Chen andheterozygote F1 females were used in the experiments. The heterozygoteswere generated by breeding male homozygotes for human CD137 in C57background together with BalbC females. All experiments were done byapproval of Malmo/Lund ethical committee.

CT26 colon cancer cells were obtained from ATCC and transfected withhuman 5T4. The CT26-5T4 cell line growing in log phase was injectedsubcutaneously (0.5×10⁶ cells in 100 μL on day 0 (D0)) into the righthind/flank. Intraperitoneal treatments (1.33 nM) were done on days 7,10, and 13.

The tumor was measured in width, length and height with a calliper, ofwhich the tumor volume was calculated (w/2×l/2×h/2×pi×(4/3)). Theanimals were terminated before the tumor volume reached 2 cm³, atwounding, or affected health of the mice.

The data were analysed for tumor volume inhibition by the bispecificantibody compared to the monoclonal antibody using GraphPad Prism andExcel.

Results and Conclusions

Anti-tumor efficacy was demonstrated using treatment with bispecificantibody 1618-1210 compared to treatment with monoclonal antibody 1618at days 8-22 in the form of tumor growth inhibition. The percentage oftumor volume inhibition ranged from 0-68% when treated with 1618-1210(see Table 22).

In conclusion, the anti-tumor effect of 1618-1210 was investigated usingtransgenic mice for human CD137 and a subcutaneous tumor model of CT26colon carcinoma transfected with human 5T4. The bispecific antibody1618-1210 demonstrated tumor volume inhibition compared to monoclonalCD137 mAb 1618.

TABLE 22 Tumor inhibition Tumor growth inhibition Day after (tumorvolume) by tumor bispecific Ab compared inoculation to monospecific AbD0  0% D6  5% D8 17% D10 43% D13 68% D15 65% D17 50% D20 45% D22 45%

Example 34 Bispecific 5T4-CD137 antibodies localize to the tumor area

Material and Methods

Female SCID-Beige mice (7-8 w) from Taconic (Denmark) were used in theexperiments. All experiments were done by approval of the Malmo/Lundethical committee.

Twin Tumor Studies (816 and CT26 Tumors)

B16.F10 wt (B16) melanoma was obtained from ATCC and cultivatedaccording to recommendations by ATCC. B16-514 was obtained fromProfessor Peter Stern and cultivated in the same medium, supplementedwith 1.2 mg/mL G418. CT26 and CT26-5T4 cells were cultivated in RPMI,10% FCS, NaPy and HEPES. CT-5T4 medium was supplemented with 1.2 mg/mLG418.

For B16 and CT26 tumors, twin tumor studies were performed and eachmouse received one 5T4 negative and one positive tumor at each side ofthe flank. The cell lines, growing in log phase, were injectedsubcutaneously (1×10⁵ cells in 100 μL at day 0). Human PBMCs (10×10⁶ in100 μL), isolated from leukocyte concentrates, were injectedintraperitoneally on the same day. Leukocyte concentrates were obtainedfrom Lund University Hospital.

Intraperitoneal antibody treatments (100 μg) were done on days 6 and 13for B16 tumors and days 6, 13 and 20 for CT26 tumors.

Single Tumor Studies (SKOV-3 Tumors)

For SKOV-3 tumors, each mouse received a single tumor in the rightflank. The cell line, growing in log phase, was injected subcutaneously(10×10⁶ cells in 100 μL on day 0). Human PBMCs (10×10⁶ in 100 μL),isolated from leukocyte concentrates, were injected intraperitoneallyonce the average tumor volume reached above 100 mm³. Intraperitonealantibody treatments (100 μg) were done starting at 6 days after the PBMCtransfer, on days 55, 62 and 67.

FACS Analysis

Mice were sacrificed 24 h after the final treatment and tumors werecollected. Tumors were enzymatically digested using Liberase TL (Roche#05401020001) and DNase I (Roche #10104159001). Digested tumor materialwas passed through a 70 μm cell strainer (Fisher Scientific #22363548)and the resulting single cell suspension was stained for FACS analysis.

Unspecific antibody binding was blocked using mouse IgG (JacksonImmunoResearch #015-000-003) and Fc block (BD #553141). Dead cells weredetected using Fixable Viability Stain 450 (BD #562247) according tomanufacturer's instructions. Binding of antibody (human IgG) to thetumor cells was detected using goat-anti-human IgG-PE (JacksonImmunoResearch #109-115-098). Samples were run on a FACSVerse (BD) anddata were analysed using FlowJo software.

Results

Localization of 1618-1210.m2.m5 to 5T4-Positive B16 and CT26 Tumors inSCID-Beige Mice

Binding of human IgG was clearly detectable on approximately 6% of thecells in B16-5T4 tumors from mice treated with 1618-1210.m2.m5, but notin B16.F10 wt tumors from the same mice. Mice treated with 1618.m2 or2112.s4.m3 had human IgG bound to <1% of cells irrespective of tumortype (FIG. 26).

A similar observation was made in CT26 tumor-bearing mice. Antibodylocalization was again observed specifically in 5T4-expressing tumors.Similar to B16 tumors, human IgG was detectable in approximately 8% ofthe viable tumor cells in mice treated with 1618-1210.m2.m5, but notwith 1618.m2 or 2112.s4.m3. Additionally, biotinylated CD137 was alsobound specifically by cells from 5T4-expressing tumors, from micetreated with 1618-1210.m2.m5 (FIG. 27).

Localization of 1618-1210.m2.m5 to SKOV-3 Tumors

Similar to what was observed for CT26 tumors, binding of biotinylatedCD137 was observed in SKOV-3 tumors from mice treated with1618-1210.m2.m5, but not 1618.m2 or 2112.s4.m3. Additionally, cells thathad bound CD137 also expressed 5T4, suggesting that the bispecificantibody targets specifically 5T4-expressing cells and remains intactwithin the tumor (FIG. 28).

Conclusion

These data show that the bispecific antibody 1618-1210.m2.m5 bindsselectively to 5T4-expressing tumors in viva In contrast, the CD137monospecific antibodies 1618.m2 and 2112.s4.m3 do not localize to thetumors.

Example 35 Enhanced Tm of Optimized 5T4 and CD137 Binders

Tm measurements were performed on soluble scFv or bispecific antibodiesusing protein fluorescence with the UNcle platform (Unchained Labs).Onset of aggregation was measured with static light scattering (SLS)with the UNcle platform. Measurements were performed in the temperaturerange 20° C.-95° C. with ramping speed of 0.4° C. per min. in PBS and ata protein concentration range of 0.12-1.32 mg/ml. The data analysis wasperformed with the UNcle Analysis software version 2.0 using defaultsettings.

As can be seen in Table 23, the optimized sequences exhibit improved Tm1and Tagg compared to the wildtype sequences (1618 and 1210,respectively).

In Table 24, the Tm1 and Tagg for exemplary bispecific antibodies (inMorrison format) is displayed. The data shows that the thermostabilityis increased when the optimized sequences are employed in the bispecificformat.

TABLE 23 scFv antibody Tm1 Tagg name Sequence ID (° C.) (° C.) 1618 16181619 56.9 51.8 1210 1210 1211 72.7 70.5 1210LO1 2992 2993 78.8 75.01618LO1 3012 3013 59.5 58.0 1618LO3 3016 3017 58.3 56.3 1618LO11 30323033 60.3 58.8 1618LO12 3034 3035 58.8 56.9 1618LO13 3036 3037 60.5 58.1

TABLE 24 Composition of construct (amino acid sequences) A B C D (VH (VL(VH (VL Additional Tm1 Antibody name of B1) of B1) of B2) of B2Connector* alterations* (° C.) Tagg (° C.) 1618LO1-1210LO1 3012 30132992 2993 m6 m2 74.7 74.5 1618LO1-1210LO2 3012 3013 2994 2995 m6 m2 73.272.6 1618LO1-1210 3012 3013 1210 1211 m6 m2 71.1 71.1 1618LO3-1210LO13016 3017 2992 2993 m6 m2 73.5 73.5 1618LO3-1210LO2 3016 3017 2994 2995m6 m2 73.0 72.8 1618LO3-1210 3016 3017 1210 1211 m6 m2 71.7 70.81618LO11-1210LO1 3032 3033 2992 2993 m6 m2 74.1 74.4 1618LO11-1210LO23032 3033 2994 2995 m6 m2 73.5 73.7 1618LO11-1210 3032 3033 1210 1211 m6m2 71.3 71.9 *See Tables D(5) and D(6) for details

REFERENCES

Akhmetzyanova, I., Zelinskyy, G., Littwitz-Salomon, E., Malyshkina, A.,Dietze, K. K., Streeck, H., Brandau, S., and Dittmer, U. (2016) CD137Agonist Therapy Can Reprogram Regulatory T Cells into Cytotoxic CD4+ TCells with Antitumor Activity. J. lmmunol. 196, 484-492.

Ascierto, P. A., Simeone, E., Sznol, M., Fu, Y. X., and Melero, I.(2010) Clinical experiences with anti-CD137 and anti-PD1 therapeuticantibodies. Semin. Oncol. 37, 508-516.

Bartkowiak, T. and Curran, M. A. (2015) 4-1BB Agonists: Multi-PotentPotentiators of Tumor Immunity. Front Oncol. 5, 117.

Boghaert, E. R., Sridharan, L., Khandke, K. M., Armellino, D., Ryan, M.G., Myers, K., Harrop, R., Kunz, A., Hamann, P. R., Marquette, K.,Dougher, M., DiJoseph, J. F., and Damle, N. K. (2008) The oncofetalprotein, 5T4, is a suitable target for antibody-guided anti-cancerchemotherapy with calicheamicin. Int J Oncol. 32, 221-234.

Castro, F. V., McGinn, O. J., Krishnan, S., Marinov, G., Li, J.,Rutkowski, A. J., Elkord, E., Burt, D. J., Holland, M., Vaghjiani, R.,Gallego, A., Saha, V., and Stern, P. L. (2012) 5T4 oncofetal antigen isexpressed in high risk of relapse childhood pre-B acute lymphoblasticleukemia and is associated with a more invasive and chemotacticphenotype. Leukemia.

Cheever, M. A., Allison, J. P., Ferris, A. S., Finn, O. J., Hastings, B.M., Hecht, T. T., Mellman, I., Prindiville, S. A., Viner, J. L., Weiner,L. M., and Matrisian, L. M. (2009) The prioritization of cancerantigens: a national cancer institute pilot project for the accelerationof translational research. Clin. Cancer Res. 15, 5323-5337.

Curran, M. A., Kim, M., Montalvo, W., Al-Shamkhani, A., and Allison, J.P. (2011) Combination CTLA-4 blockade and 4-1BB activation enhancestumor rejection by increasing T-cell infiltration, proliferation, andcytokine production. PLoS. ONE. 6, e19499.

Damelin, M., Geles, K. G., Follettie, M. T., Yuan, P., Baxter, M.,Golas, J., DiJoseph, J. F., Karnoub, M., Huang, S., Diesl, V., Behrens,C., Choe, S. E., Rios, C., Gruzas, J., Sridharan, L., Dougher, M., Kunz,A., Hamann, P. R., Evans, D., Armellino, D., Khandke, K., Marquette, K.,Tchistiakova, L., Boghaert, E. R., Abraham, R. T., Wistuba, I. I., andZhou, B. B. (2011) Delineation of a cellular hierarchy in lung cancerreveals an oncofetal antigen expressed on tumor-initiating cells. CancerRes 71, 4236-4246.

Dubrot, J., Milheiro, F., Alfaro, C., Palazon, A., Martinez-Forero, I.,Perez-Gracia, J. L., Morales-Kastresana, A., Romero-Trevejo, J. L.,Ochoa, M. C., Hervas-Stubbs, S., Prieto, J., Jure-Kunkel, M., Chen, L.,and Melero, I. (2010) Treatment with anti-CD137 mAbs causes intenseaccumulations of liver T cells without selective antitumorimmunotherapeutic effects in this organ. Cancer Immunol. lmmunother. 59,1223-1233.

Elkord, E., Shablak, A., Stern, P. L., and Hawkins, R. E. (2009) 5T4 asa target for immunotherapy in renal cell carcinoma. Expert RevAnticancer Ther 9, 1705-1709.

Forsberg, G., Ohlsson, L., Brodin, T., Bjork, P., Lando, P. A., Shaw,D., Stern, P. L., and Dohlsten, M. (2001) Therapy of humannon-small-cell lung carcinoma using antibody targeting of a modifiedsuperantigen. Br. J Cancer 85, 129-136.

Garber, K. (2011) Beyond ipilimumab: new approaches target theimmunological synapse. J Natl Cancer Inst. 103, 1079-1082.

Gauttier, V., Judor, J. P., Le, G., V, Cany, J., Ferry, N., and Conchon,S. (2014) Agonistic anti-CD137 antibody treatment leads to antitumorresponse in mice with liver cancer. Int. J. Cancer 135, 2857-2867.

Gray, J. C., French, R. R., James, S., Al-Shamkhani, A., Johnson, P. W.,and Glennie, M. J. (2008) Optimising anti-tumour CD8 T-cell responsesusing combinations of immunomodulatory antibodies. Eur. J. Immunol. 38,2499-2511.

Guo, Z., Cheng, D., Xia, Z., Luan, M., Wu, L., Wang, G., and Zhang, S.(2013) Combined TIM-3 blockade and CD137 activation affords thelong-term protection in a murine model of ovarian cancer. J. Transl.Med. 11, 215.

Hole, N. and Stern, P. L. (1988) A 72 kD trophoblast glycoproteindefined by a monoclonal antibody. Br. J Cancer 57, 239-246.

Hornig, N., Reinhardt, K., Kermer, V., Kontermann, R. E., and Muller, D.(2013) Evaluating combinations of costimulatory antibody-ligand fusionproteins for targeted cancer immunotherapy. Cancer Immunol Immunother.

Kermer, V., Hornig, N., Harder, M., Bondarieva, A., Kontermann, R. E.,and Muller, D. (2014) Combining antibody-directed presentation of IL-15and 4-1BBL in a trifunctional fusion protein for cancer immunotherapy.Mol. Cancer Ther. 13, 112-121.

Kiefer, J. D. and Neri, D. (2016) Immunocytokines and bispecificantibodies: two complementary strategies for the selective activation ofimmune cells at the tumor site. Immunol. Rev. 270, 178-192.

Kim, J. A., Averbook, B. J., Chambers, K., Rothchild, K., Kjaergaard,J., Papay, R., and Shu, S. (2001) Divergent effects of 4-1BB antibodieson antitumor immunity and on tumor-reactive T-cell generation. CancerRes 61, 2031-2037.

Kwong, B., Gai, S. A., Elkhader, J., Wittrup, K. D., and Irvine, D. J.(2013) Localized immunotherapy via liposome-anchored Anti-CD137+IL-2prevents lethal toxicity and elicits local and systemic antitumorimmunity. Cancer Res. 73, 1547-1558.

Lee, H. W., Park, S. J., Choi, B. K., Kim, H. H., Nam, K. O., and Kwon,B. S. (2002) 4-1BB promotes the survival of CD8+T lymphocytes byincreasing expression of Bcl-xL and Bfl-1. J Immunol 169, 4882-4888.

Lee, S. J., Myers, L., Muralimohan, G., Dai, J., Qiao, Y., Li, Z.,Mittler, R. S., and Vella, A. T. (2004) 4-1BB and OX40 dualcostimulation synergistically stimulate primary specific CD8 T cells forrobust effector function. J. Immunol. 173, 3002-3012.

Li, F. and Ravetch, J. V. (2011) Inhibitory Fcgamma receptor engagementdrives adjuvant and anti-tumor activities of agonistic CD40 antibodies.Science 333, 1030-1034.

Li, S. Y. and Liu, Y. (2013) Immunotherapy of melanoma with the immunecostimulatory monoclonal antibodies targeting CD137. Clin. Pharmacol. 5,47-53.

Liu, R., Jiang, W., Yang, M., Guo, H., Zhang, Y., Wang, J., Zhu, H.,Shi, R., Fan, D., Yang, C., Zhu, Z., Xie, Y., and Xiong, D. (2010)Efficient inhibition of human B-cell lymphoma in SCID mice bysynergistic antitumor effect of human 4-1BB ligand/anti-CD20 fusionproteins and anti-CD3/anti-CD20 diabodies. J. Immunother. 33, 500-509.

McMillin, D. W., Hewes, B., Gangadharan, B., Archer, D. R., Mittler, R.S., and Spencer, H. T. (2006) Complete regression of large solid tumorsusing engineered drug-resistant hematopoietic cells and anti-CD137immunotherapy. Hum. Gene Ther 17, 798-806.

Melero, I., Shuford, W. W., Newby, S. A., Aruffo, A., Ledbetter, J. A.,Hellstrom, K. E., Mittler, R. S., and Chen, L. (1997) Monoclonalantibodies against the 4-1BB T-cell activation molecule eradicateestablished tumors. Nat Med 3, 682-685.

Miller, R. E., Jones, J., Le, T., Whitmore, J., Boiani, N., Gliniak, B.,and Lynch, D. H. (2002) 4-1BB-specific monoclonal antibody promotes thegeneration of tumor-specific immune responses by direct activation ofCD8 T cells in a CD40-dependent manner. J Immunol 169, 1792-1800.

Morales-Kastresana, A., Sanmamed, M. F., Rodriguez, I., Palazon, A.,Martinez-Forero, I., Labiano, S., Hervas-Stubbs, S., Sangro, B., Ochoa,C., Rouzaut, A., Azpilikueta, A., Bolanos, E., Jure-Kunkel, M.,Gutgemann, I., and Melero, I. (2013) Combined immunostimulatorymonoclonal antibodies extend survival in an aggressive transgenichepatocellular carcinoma mouse model. Clin. Cancer Res. 19, 6151-6162.

Niu, L., Strahotin, S., Hewes, B., Zhang, B., Zhang, Y., Archer, D.,Spencer, T., Dillehay, D., Kwon, B., Chen, L., Vella, A. T., andMittler, R. S. (2007) Cytokine-mediated disruption of lymphocytetrafficking, hemopoiesis, and induction of lymphopenia, anemia, andthrombocytopenia in anti-CD137-treated mice. J. Immunol. 178, 4194-4213.

Pan, P. Y., Zang, Y., Weber, K., Meseck, M. L., and Chen, S. H. (2002)OX40 ligation enhances primary and memory cytotoxic T lymphocyteresponses in an immunotherapy for hepatic colon metastases. Mol Ther 6,528-536.

Pastor, F., Kolonias, D., McNamara, J. O., and Gilboa, E. (2011)Targeting 4-1BB costimulation to disseminated tumor lesions withbi-specific oligonucleotide aptamers. Mol Ther 19, 1878-1886.

Pulle, G., Vidric, M., and Watts, T. H. (2006) IL-15-dependent inductionof 4-1BB promotes antigen-independent CD8 memory T cell survival. JImmunol 176, 2739-2748.

Rabu, C., Quemener, A., Jacques, Y., Echasserieau, K., Vusio, P., andLang, F. (2005) Production of recombinant human trimeric CD137L(4-1BBL). Cross-linking is essential to its T cell co-stimulationactivity. J Biol Chem 280, 41472-41481.

Sallin, M. A., Zhang, X., So, E. C., Burch, E., Cai, L., Lin, W.,Chapoval, A. I., and Strome, S. E. (2014) The anti-lymphoma activitiesof anti-CD137 monoclonal antibodies are enhanced in FcgammaRIII(−/−)mice. Cancer Immunol. Immunother. 63, 947-958.

Sanmamed, M. F., Pastor, F., Rodriguez, A., Perez-Gracia, J. L.,Rodriguez-Ruiz, M. E., Jure-Kunkel, M., and Melero, I. (2015) Agonistsof Co-stimulation in Cancer Immunotherapy Directed Against CD137, OX40,GITR, CD27, CD28, and ICOS. Semin. Oncol. 42, 640-655.

Schrand, B., Berezhnoy, A., Brenneman, R., Williams, A., Levay, A.,Kong, L. Y., Rao, G., Zhou, S., Heimberger, A. B., and Gilboa, E. (2014)Targeting 4-1BB costimulation to the tumor stroma with bispecificaptamer conjugates enhances the therapeutic index of tumorimmunotherapy. Cancer Immunol. Res. 2, 867-877.

Shuford, W. W., Klussman, K., Tritchler, D. D., Loo, D. T., Chalupny,J., Siadak, A. W., Brown, T. J., Emswiler, J., Raecho, H., Larsen, C.P., Pearson, T. C., Ledbetter, J. A., Aruffo, A., and Mittler, R. S.(1997) 4-1BB costimulatory signals preferentially induce CD8+ T cellproliferation and lead to the amplification in vivo of cytotoxic T cellresponses. J Exp. Med 186, 47-55.

So, T., Lee, S. W., and Croft, M. (2008) Immune regulation and controlof regulatory T cells by OX40 and 4-1BB. Cytokine Growth Factor Rev. 19,253-262.

Southall, P. J., Boxer, G. M., Bagshawe, K. D., Hole, N., Bromley, M.,and Stern, P. L. (1990) Immunohistological distribution of 5T4 antigenin normal and malignant tissues. Br. J Cancer 61, 89-95.

Southgate, T. D., McGinn, O. J., Castro, F. V., Rutkowski, A. J.,Al-Muftah, M., Marinov, G., Smethurst, G. J., Shaw, D., Ward, C. M.,Miller, C. J., and Stern, P. L. (2010) CXCR4 mediated chemotaxis isregulated by 5T4 oncofetal glycoprotein in mouse embryonic cells. PLoS.ONE. 5, e9982.

St Rose, M. C., Taylor, R. A., Bandyopadhyay, S., Qui, H. Z., Hagymasi,A. T., Vella, A. T., and Adler, A. J. (2013) CD134/CD137 dualcostimulation-elicited IFN-gamma maximizes effector T-cell function butlimits Treg expansion. Immunol. Cell Biol. 91, 173-183.

Taraban, V. Y., Rowley, T. F., O'Brien, L., Chan, H. T., Haswell, L. E.,Green, M. H., Tutt, A. L., Glennie, M. J., and Al-Shamkhani, A. (2002)Expression and costimulatory effects of the TNF receptor superfamilymembers CD134 (OX40) and CD137 (4-1BB), and their role in the generationof anti-tumor immune responses. Eur J Immunol 32, 3617-3627.

Uno, T., Takeda, K., Kojima, Y., Yoshizawa, H., Akiba, H., Mittler, R.S., Gejyo, F., Okumura, K., Yagita, H., and Smyth, M. J. (2006)Eradication of established tumors in mice by a combinationantibody-based therapy. Nat. Med. 12, 693-698.

Vinay, D. S. and Kwon, B. S. (2012) Immunotherapy of cancer with 4-1BB.Mol. Cancer Ther. 11, 1062-1070.

Wei, H., Zhao, L., Li, W., Fan, K., Qian, W., Hou, S., Wang, H., Dai,M., Hellstrom, I., Hellstrom, K. E., and Guo, Y. (2013) CombinatorialPD-1 blockade and CD137 activation has therapeutic efficacy in murinecancer models and synergizes with cisplatin. PLoS. ONE. 8, e84927.

Westwood, J. A., Darcy, P. K., Guru, P. M., Sharkey, J., Pegram, H. J.,Amos, S. M., Smyth, M. J., and Kershaw, M. H. (2010) Three agonistantibodies in combination with high-dose IL-2 eradicate orthotopickidney cancer in mice. J. Transl. Med. 8, 42.

Westwood, J. A., Matthews, G. M., Shortt, J., Faulkner, D., Pegram, H.J., Duong, C. P., Chesi, M., Bergsagel, P. L., Sharp, L. L., Huhn, R.D., Darcy, P. K., Johnstone, R. W., and Kershaw, M. H. (2014a)Combination anti-CD137 and anti-CD40 antibody therapy in murinemyc-driven hematological cancers. Leuk. Res. 38, 948-954.

Westwood, J. A., Potdevin Hunnam, T. C., Pegram, H. J., Hicks, R. J.,Darcy, P. K., and Kershaw, M. H. (2014b) Routes of delivery for CpG andanti-CD137 for the treatment of orthotopic kidney tumors in mice. PLoS.ONE. 9, e95847.

White, A. L., Chan, H. T., French, R. R., Beers, S. A., Cragg, M. S.,Johnson, P. W., and Glennie, M. J. (2013) FcgammaRlotalotaB controls thepotency of agonistic anti-TNFR mAbs. Cancer Immunol Immunother 62,941-948.

White, A. L., Chan, H. T., Roghanian, A., French, R. R., Mockridge, C.I., Tutt, A. L., Dixon, S. V., Ajona, D., Verbeek, J. S., Al-Shamkhani,A., Cragg, M. S., Beers, S. A., and Glennie, M. J. (2011) Interactionwith Fc{gamma}RIIB Is Critical for the Agonistic Activity of Anti-CD40Monoclonal Antibody. J Immunol 187, 1754-1763.

Wilcox, R. A., Flies, D. B., Zhu, G., Johnson, A. J., Tamada, K.,Chapoval, A. I., Strome, S. E., Pease, L. R., and Chen, L. (2002)Provision of antigen and CD137 signaling breaks immunological ignorance,promoting regression of poorly immunogenic tumors. J Clin Invest 109,651-659.

Wilson, N. S., Yang, B., Yang, A., Loeser, S., Marsters, S., Lawrence,D., Li, Y., Pitti, R., Totpal, K., Yee, S., Ross, S., Vernes, J. M., Lu,Y., Adams, C., Offringa, R., Kelley, B., Hymowitz, S., Daniel, D., Meng,G., and Ashkenazi, A. (2011b) An Fcgamma receptor-dependent mechanismdrives antibody-mediated target-receptor signaling in cancer cells.Cancer Cell 19, 101-113.

Wilson, N. S., Yang, B., Yang, A., Loeser, S., Marsters, S., Lawrence,D., Li, Y., Pitti, R., Totpal, K., Yee, S., Ross, S., Vernes, J. M., Lu,Y., Adams, C., Offringa, R., Kelley, B., Hymowitz, S., Daniel, D., Meng,G., and Ashkenazi, A. (2011a) An Fcgamma receptor-dependent mechanismdrives antibody-mediated target-receptor signaling in cancer cells.Cancer Cell 19, 101-113.

Wyzgol, A., Muller, N., Fick, A., Munkel, S., Grigoleit, G. U.,Pfizenmaier, K., and Wajant, H. (2009) Trimer stabilization,oligomerization, and antibody-mediated cell surface immobilizationimprove the activity of soluble trimers of CD27L, CD4OL, 41BBL, andglucocorticoid-induced TNF receptor ligand. J Immunol 183, 1851-1861.

Ye, Q., Song, D. G., Poussin, M., Yamamoto, T., Best, A., Li, C.,Coukos, G., and Powell, D. J., Jr. (2014) CD137 accurately identifiesand enriches for naturally occurring tumor-reactive T cells in tumor.Clin Cancer Res 20, 44-55.

Zhang, N., Sadun, R. E., Arias, R. S., Flanagan, M. L., Sachsman, S. M.,Nien, Y. C., Khawli, L. A., Hu, P., and Epstein, A. L. (2007) Targetedand untargeted CD137L fusion proteins for the immunotherapy ofexperimental solid tumors. Clin. Cancer Res. 13, 2758-2767.

1. A bispecific polypeptide comprising a first binding domain,designated B1, which is capable of binding specifically to CD137, and asecond binding domain, designated B2, which is capable of specificallybinding to a tumour cell-associated antigen.
 2. A polypeptide accordingto claim 1, wherein the first and/or second binding domains are/isselected from the group consisting of antibodies and antigen-bindingfragments thereof.
 3. A polypeptide according to claim 2 wherein theantigen-binding fragment is selected from the group consisting of: Fvfragments (such as a single chain Fv fragment, or a disulphide-bonded Fvfragment), Fab-like fragments (such as a Fab fragment; a Fab′ fragmentor a F(ab)₂ fragment) and domain antibodies.
 4. A polypeptide accordingto any one of the preceding claims wherein the polypeptide is abispecific antibody.
 5. A polypeptide according to claim 4 wherein: (a)binding domain B1 and/or binding domain B2 is an intact IgG antibody;(b) binding domain B1 and/or binding domain B2 is an Fv fragment; (c)binding domain B1 and/or binding domain B2 is a Fab fragment; and/or (d)binding domain B1 and/or binding domain B2 is a single domain antibody.6. A polypeptide according to claim 4 or 5 wherein the bispecificantibody comprises a human Fc region or a variant of a said region,where the region is an IgG1, IgG2, IgG3 or IgG4 region, preferably anIgG1 or IgG4 Fc region.
 7. A polypeptide according to claim 6 whereinthe Fc exhibits no or very low affinity for FcgR.
 8. A polypeptideaccording to claim 6 or 7 wherein the Fc region is a variant of a humanIgG1 Fc region comprising a mutation at one or more of the followingpositions: L234, L235, P239, D265, N297 and/or P329.
 9. A polypeptideaccording to claim 8 wherein alanine is present at the mutatedpositions(s).
 10. A polypeptide according to claim 9 wherein the Fcregion is a variant of a human IgG1 Fc region comprising the doublemutations L234A and L235A.
 11. A polypeptide according to any one ofclaims 4 to 10 wherein the bispecific antibody is selected from thegroups consisting of: (a) bivalent bispecific antibodies, such asIgG-scFv bispecific antibodies (for example, wherein B1 is an intact IgGand B2 is an scFv attached to B1 at the N-terminus of a light chainand/or at the C-terminus of a light chain and/or at the N-terminus of aheavy chain and/or at the C-terminus of a heavy chain of the IgG, orvice versa); (b) monovalent bispecific antibodies, such as a DuoBody® ora ‘knob-in-hole’ bispecific antibody (for example, an scFv-KIH,scFv-KIHr, a BiTE-KIH or a BiTE-KIHr; (c) scFv₂-Fc bispecific antibodies(for example, ADAPTIR™ bispecific antibodies); (d) BITE/scFv2 bispecificantibodies; (e) DVD-Ig bispecific antibodies or other IgG-FAb, FAb-IgGbispecific antibodies regardless of bivalency or linkers/connectorsemployed; (f) DART-based bispecific antibodies (for example, DART₂-Fc,DART₂-Fc or DART); (g) DNL-Fabs bispecific antibodies; and (h)scFv-HSA-scFv bispecific antibodies.
 12. A polypeptide according toclaim 11 wherein the bispecific antibody is an IgG-scFv bispecificantibody.
 13. A polypeptide according to any one of the preceding claimswherein binding domain B1 and binding domain B2 are fused directly toeach other.
 14. A polypeptide according to any one of claims 1 to 12wherein binding domain B1 and binding domain B2 are joined via apolypeptide linker.
 15. A polypeptide according to claim 14 wherein thelinker is selected from the group consisting of the amino acid sequenceSGGGGSGGGGS (SEQ ID NO: 87), SGGGGSGGGGSAP (SEQ ID NO: 88), NFSQP (SEQID NO: 89), KRTVA (SEQ ID NO: 90), GGGSGGGG (SEQ ID NO: 91), GGGGSGGGGS(SEQ ID NO: 92), GGGGSGGGGSGGGGS (SEQ ID NO: 93), THTCPPCPEPKSSDK (SEQID NO: 140), GGGS (SEQ ID NO: 141), EAAKEAAKGGGGS (SEQ ID NO: 142),EAAKEAAK (SEQ ID NO: 143), or (SG)m, where m=1 to
 7. 16. A polypeptideaccording to any of the preceding claims, wherein the polypeptide isincapable of inducing antibody dependent cell cytotoxicity (ADCC),antibody-dependent cellular phagocytosis (ADCP) and/orcomplement-dependent cytotoxicity (CDC).
 17. A polypeptide according toany of the preceding claims, wherein the polypeptide is capable ofinducing tumour immunity.
 18. A polypeptide according to any one of thepreceding claims, wherein the polypeptide is capable of inducing: (a)activation of cytotoxic T cells, i.e. CD8+ T cells; (b) activation ofhelper T cells, i.e. CD4⁺ T cells; (c) activation of dendritic cells;and/or (d) activation of natural killer cells; and/or (e) reprogramingof Tregs into effector T cells.
 19. A polypeptide according to any ofthe preceding claims wherein binding domain B1 binds to human CD137 witha K_(D) of less than 10×10⁻⁹M, for example less than 4×10⁻⁹M or lessthan 1.2×10⁻⁹M.
 20. A polypeptide according to any of the precedingclaims wherein binding domain B1 comprises: (a) the three CDRs of theheavy chain and/or the three CDRs of the light chain of antibody1200/1201 (SEQ ID NOs: 54, 55 and 79 and/or SEQ ID NOs: 46, 65 and 72);(b) the three CDRs of the heavy chain and/or the three CDRs of the lightchain of antibody 1202/1203 (SEQ ID NOs: 54, 55 and 80 and/or SEQ IDNOs: 60, 66 and 73); (c) the three CDRs of the heavy chain and/or thethree CDRs of the light chain of antibody 1204/1205 (SEQ ID NOs: 54, 55and 81 and/or SEQ ID NOs: 61, 67, 74); (d) the three CDRs of the heavychain and/or the three CDRs of the light chain of antibody 1214/1215(SEQ ID NOs: 54, 55 and 82 and/or SEQ ID NOs: 46, 68 and 75); (e) thethree CDRs of the heavy chain and/or the three CDRs of the light chainof antibody 161⁸/₁619 (SEQ ID NOs: 54, 55 and 83 and/or SEQ ID NOs: 62,69 and 76); (f) the three CDRs of the heavy chain and/or the three CDRsof the light chain of antibody 1620/1621 (SEQ ID NOs: 54, 55 and 84and/or SEQ ID NOs: 63, 70, and 77); (g) the three CDRs of the heavychain and/or the three CDRs of the light chain of antibody 1626/1627(SEQ ID NOs: 54, 55 and 85 and/or SEQ ID NOs: 64, 71 and 78); (h) thethree CDRs of the heavy chain and/or the three CDRs of the light chainof antibody 3012/3013 (SEQ ID NOs: 156, 69 and 76 and/or SEQ ID NOs:158, 155 and 83); (i) the three CDRs of the heavy chain and/or the threeCDRs of the light chain of antibody 3014/3015 (SEQ ID NOs: 62, 69 and 76and/or SEQ ID NOs: 159, 160 and 83); (j) the three CDRs of the heavychain and/or the three CDRs of the light chain of antibody 3016/3017(SEQ ID NOs: 62, 69 and 76 and/or SEQ ID NOs: 159, 155 and 83); (k) thethree CDRs of the heavy chain and/or the three CDRs of the light chainof antibody 3018/3019 (SEQ ID NOs: 156, 69 and 76 and/or SEQ ID NOs:158, 161 and 83); (l) the three CDRs of the heavy chain and/or the threeCDRs of the light chain of antibody 3020/3021 (SEQ ID NOs: 156, 69 and76 and/or SEQ ID NOs: 162, 163 and 83); (m) the three CDRs of the heavychain and/or the three CDRs of the light chain of antibody 3022/3023(SEQ ID NOs: 156, 69 and 76 and/or SEQ ID NOs: 159, 155 and 83); (n) thethree CDRs of the heavy chain and/or the three CDRs of the light chainof antibody 3024/3025 (SEQ ID NOs: 156, 69 and 76 and/or SEQ ID NOs: 54,55 and 83); (o) the three CDRs of the heavy chain and/or the three CDRsof the light chain of antibody 3026/3027 (SEQ ID NOs: 156, 69 and 76and/or SEQ ID NOs: 162, 165 and 83); (p) the three CDRs of the heavychain and/or the three CDRs of the light chain of antibody 3028/3029(SEQ ID NOs: 157, 69 and 76 and/or SEQ ID NOs: 159, 166 and 83); (q) thethree CDRs of the heavy chain and/or the three CDRs of the light chainof antibody 3030/3031 (SEQ ID NOs: 156, 69 and 76 and/or SEQ ID NOs: 54,166 and 83); (r) the three CDRs of the heavy chain and/or the three CDRsof the light chain of antibody 3032/3033 (SEQ ID NOs: 156, 69 and 76and/or SEQ ID NOs: 54, 55 and 83); (s) the three CDRs of the heavy chainand/or the three CDRs of the light chain of antibody 303⁴/₃035 (SEQ IDNOs: 62, 69 and 76 and/or SEQ ID NOs: 54, 155 and 83); or (t) the threeCDRs of the heavy chain and/or the three CDRs of the light chain ofantibody 3036/3037 (SEQ ID NOs: 156, 69 and 76 and/or SEQ ID NOs: 162,155 and 83).
 21. A polypeptide according to any of the preceding claimswherein binding domain B1 comprises: (a) the heavy chain variable regionand/or the light chain variable region of antibody 1200/1201 (SEQ ID NO:19 and/or SEQ ID NO: 17); (b) the heavy chain variable region and/or thelight chain variable region of antibody 1202/1203 (SEQ ID NO: 23 and/orSEQ ID NO: 21); (c) the heavy chain variable region and/or the lightchain variable region of antibody 1204/1205 (SEQ ID NO: 25 and/or SEQ IDNO: 27); (d) the heavy chain variable region and/or the light chainvariable region of antibody 1214/1215 (SEQ ID NO: 31 and/or SEQ ID NO:29); (e) the heavy chain variable region and/or the light chain variableregion of antibody 1618/1619 (SEQ ID NO: 35 and/or SEQ ID NO: 33); (f)the heavy chain variable region and/or the light chain variable regionof antibody 1620/1621 (SEQ ID NO: 39 and/or SEQ ID NO: 37); (g) theheavy chain variable region and/or the light chain variable region ofantibody 1626/1627 (SEQ ID NO: 43 and/or SEQ ID NO: 41); (h) the heavychain variable region and/or the light chain variable region of antibody3012/3013 (SEQ ID NO: 114 and/or SEQ ID NO: 115); (i) the heavy chainvariable region and/or the light chain variable region of antibody3014/3015 (SEQ ID NO: 116 and/or SEQ ID NO: 117); (j) the heavy chainvariable region and/or the light chain variable region of antibody3016/3017 (SEQ ID NO: 118 and/or SEQ ID NO: 119); (k) the heavy chainvariable region and/or the light chain variable region of antibody3018/3019 (SEQ ID NO: 120 and/or SEQ ID NO: 121); (l) the heavy chainvariable region and/or the light chain variable region of antibody3020/3021 (SEQ ID NO: 122 and/or SEQ ID NO: 123); (m)the heavy chainvariable region and/or the light chain variable region of antibody3022/3023 (SEQ ID NO: 124 and/or SEQ ID NO: 125); (n) the heavy chainvariable region and/or the light chain variable region of antibody3024/3025 (SEQ ID NO: 126 and/or SEQ ID NO: 127); (o) the heavy chainvariable region and/or the light chain variable region of antibody3026/3027 (SEQ ID NO: 128 and/or SEQ ID NO: 129); (p) the heavy chainvariable region and/or the light chain variable region of antibody3028/3029 (SEQ ID NO: 130 and/or SEQ ID NO: 131); (q) the heavy chainvariable region and/or the light chain variable region of antibody3030/3031 (SEQ ID NO: 132 and/or SEQ ID NO: 133); (r) the heavy chainvariable region and/or the light chain variable region of antibody3032/3033 (SEQ ID NO: 134 and/or SEQ ID NO: 135); (s) the heavy chainvariable region and/or the light chain variable region of antibody3034/3035 (SEQ ID NO: 136 and/or SEQ ID NO: 137); or (t) the heavy chainvariable region and/or the light chain variable region of antibody3036/3037 (SEQ ID NO: 138 and/or SEQ ID NO: 139).
 22. A polypeptideaccording to any of the preceding claims wherein binding domain B1comprises: (a) the heavy chain and/or the light chain of antibody1200/1201; (b) the heavy chain and/or the light chain of antibody1202/1203; (c) the heavy chain and/or the light chain of antibody1204/1205; (d) the heavy chain and/or the light chain of antibody1214/1215; (e) the heavy chain and/or the light chain of antibody1618/1619; (f) the heavy chain and/or the light chain of antibody1620/1621; (g) the heavy chain and/or the light chain of antibody1626/1627; (h) the heavy chain and/or the light chain of antibody3012/3013; (i) the heavy chain and/or the light chain of antibody3014/3015; (j) the heavy chain and/or the light chain of antibody3016/3017; (k) the heavy chain and/or the light chain of antibody3018/3019; (l) the heavy chain and/or the light chain of antibody3020/3021; (m)the heavy chain and/or the light chain of antibody3022/3023; (n) the heavy chain and/or the light chain of antibody3024/3025; (o) the heavy chain and/or the light chain of antibody3026/3027; (p) the heavy chain and/or the light chain of antibody3028/3029; (q) the heavy chain and/or the light chain of antibody3030/3031; (r) the heavy chain and/or the light chain of antibody3032/3033; (s) the heavy chain and/or the light chain of antibody3034/3035; or (t) the heavy chain and/or the light chain of antibody3036/3037.
 23. A polypeptide according to any of the preceding claimswherein binding domain B1 comprises the light chain variable region andthe heavy chain variable region of antibody 1200/1201 (SEQ ID NO: 19and/or SEQ ID NO: 17), or a variant which has more than 60%, or morethan 70%, e.g. 75 or 80%, preferably more than 85%, e.g. more than 90 or95% amino acid identity to SEQ ID NO: 19 and/or SEQ ID NO: 17).
 24. Apolypeptide according to any one of claims 1 to 22 wherein bindingdomain B1 comprises the light chain variable region and the heavy chainvariable region of antibody 1618/1619 (SEQ ID NO: 35 and/or SEQ ID NO:33), ora variant which has more than 60%, or more than 70%, e.g. 75 or80%, preferably more than 85%, e.g. more than 90 or 95% amino acididentity to SEQ ID NO: 35 and/or SEQ ID NO: 33).
 25. A polypeptideaccording to any of the preceding claims wherein binding domain B2 bindsto a tumour cell-associated antigen selected from the group consistingof: (a) products of mutated oncogenes and tumour suppressor genes; (b)overexpressed or aberrantly expressed cellular proteins; (c) tumourantigens produced by oncogenic viruses; (d) oncofetal antigens; (e)altered cell surface glycolipids and glycoproteins; (f) celltype-specific differentiation antigens; (g) hypoxia-induced antigens;(h) tumour peptides presented by MHC class I; (i) epithelial tumourantigens; (j) haematological tumour-associated antigens; (k) cancertestis antigens; and (l) melanoma antigens.
 26. A polypeptide accordingto any of the preceding claims wherein the tumour cell-associatedantigen is selected from the group consisting of 5T4, CD20, CD19, MUC-1,carcinoembryonic antigen (CEA), CA-125, C017-1A, EpCAM, HER2, EGFR,HER3, GD2, Podocalyxin, TROP-2, DLK-1, Ox1R, Nectin-4, FAP, EphA2,EphA3, mesothelin, E-cadherin, CD24 and VEGFR.
 27. A polypeptideaccording to any of the preceding claims wherein the tumourcell-associated antigen is an oncofetal antigen.
 28. A polypeptideaccording to any of the preceding claims wherein the tumourcell-associated antigen is 5T4.
 29. A polypeptide according to any ofthe preceding claims wherein the tumour cell is a solid tumour cell. 30.A polypeptide according to claim 29 wherein the solid tumour is selectedfrom the groups consisting of renal cell carcinoma, colorectal cancer,lung cancer, prostate cancer, breast cancer, melanomas, bladder cancer,brain/CNS cancer, cervical cancer, oesophageal cancer, gastric cancer,head/neck cancer, kidney cancer, liver cancer, lymphomas, ovariancancer, pancreatic cancer and sarcomas.
 31. A polypeptide according toany of the preceding claims wherein binding domain B2 binds to thetumour cell-associated antigen with a KD of less than 10×10⁻⁹M, forexample less than 4×10⁻⁹M or less than 1.2×10⁻⁹M.
 32. A polypeptideaccording to any of the preceding claims wherein binding domain B2comprises: (a) the three CDRs of the heavy chain and/or the three CDRsof the light chain of antibody 1206/1207 (SEQ ID NOs: 54, 55 and 56and/or SEQ ID NOs: 45, 47 and 50); (b) the three CDRs of the heavy chainand/or the three CDRs of the light chain of antibody 1208/1135 (SEQ IDNOs: 54, 55 and 57 and/or SEQ ID NOs: 46, 48 and 51); (c) the three CDRsof the heavy chain and/or the three CDRs of the light chain of antibody1210/1211 (SEQ ID NOs: 54, 55 and 58 and/or SEQ ID NOs: 46, 48 and 52);(d) the three CDRs of the heavy chain and/or the three CDRs of the lightchain of antibody 1212/1213 (SEQ ID NOs: 54, 55 and 59 and/or SEQ IDNOs: 46, 49 and 53); (e) the three CDRs of the heavy chain and/or thethree CDRs of the light chain of antibody 2992/2993 (SEQ ID NOs: 144, 48and 52 and/or SEQ ID NOs: 145, 55 and 58); (f) the three CDRs of theheavy chain and/or the three CDRs of the light chain of antibody2994/2995 (SEQ ID NOs: 146, 147 and 52 and/or SEQ ID NOs: 145, 55 and58); (g) the three CDRs of the heavy chain and/or the three CDRs of thelight chain of antibody 2996/2997 (SEQ ID NOs: 146, 48 and 52 and/or SEQID NOs: 148, 55 and 58); (h) the three CDRs of the heavy chain and/orthe three CDRs of the light chain of antibody 2998/2999 (SEQ ID NOs:146, 48 and 52 and/or SEQ ID NOs: 149, 55 and 58); (i) the three CDRs ofthe heavy chain and/or the three CDRs of the light chain of antibody3000/3001 (SEQ ID NOs: 150, 48 and 52 and/or SEQ ID NOs: 148, 151 and58); (j) the three CDRs of the heavy chain and/or the three CDRs of thelight chain of antibody 3002/3003 (SEQ ID NOs: 152, 48 and 52 and/or SEQID NOs: 145, 55 and 58); (k) the three CDRs of the heavy chain and/orthe three CDRs of the light chain of antibody 3004/3005 (SEQ ID NOs:146, 48 and 52 and/or SEQ ID NOs: 153, 55 and 58); (l) the three CDRs ofthe heavy chain and/or the three CDRs of the light chain of antibody3006/3007 (SEQ ID NOs: 144, 48 and 52 and/or SEQ ID NOs: 154, 155 and58); or (m)the three CDRs of the heavy chain and/or the three CDRs ofthe light chain of antibody 3008/3009 (SEQ ID NOs: 146, 48 and 52 and/orSEQ ID NOs: 154, 55 and 58).
 33. A polypeptide according to any of thepreceding claims wherein binding domain B2 comprises: (a) the heavychain variable region and/or the light chain variable region of antibody1206/1207 (SEQ ID NO: 3 and/or SEQ ID NO: 1); (b) the heavy chainvariable region and/or the light chain variable region of antibody1208/1135 (SEQ ID NO: 7 and/or SEQ ID NO: 5); (c) the heavy chainvariable region and/or the light chain variable region of antibody1210/1211 (SEQ ID NO: 11 and/or SEQ ID NO: 9); (d) the heavy chainvariable region and/or the light chain variable region of antibody1212/1213 (SEQ ID NO: 15 and/or SEQ ID NO: 13); (e) the heavy chainvariable region and/or the light chain variable region of antibody2992/2993 (SEQ ID NO: 96 and/or SEQ ID NO: 97); (f) the heavy chainvariable region and/or the light chain variable region of antibody2994/2995 (SEQ ID NO: 98 and/or SEQ ID NO: 99); (g) the heavy chainvariable region and/or the light chain variable region of antibody2996/2997 (SEQ ID NO: 100 and/or SEQ ID NO: 101); (h) the heavy chainvariable region and/or the light chain variable region of antibody2998/2999 (SEQ ID NO: 102 and/or SEQ ID NO: 103); (i) the heavy chainvariable region and/or the light chain variable region of antibody3000/3001 (SEQ ID NO: 104 and/or SEQ ID NO: 105); (j) the heavy chainvariable region and/or the light chain variable region of antibody3002/3003 (SEQ ID NO: 106 and/or SEQ ID NO: 107); (k) the heavy chainvariable region and/or the light chain variable region of antibody3004/3005 (SEQ ID NO: 108 and/or SEQ ID NO: 109); (l) the heavy chainvariable region and/or the light chain variable region of antibody3006/3007 (SEQ ID NO: 110 and/or SEQ ID NO: 111); or (m)the heavy chainvariable region and/or the light chain variable region of antibody3008/3009 (SEQ ID NO: 112 and/or SEQ ID NO: 113).
 34. A polypeptideaccording to any of the preceding claims wherein binding domain B2comprises: (a) the heavy chain and/or the light chain of antibody1206/1207; (b) the heavy chain and/or the light chain of antibody1208/1135; (c) the heavy chain and/or the light chain of antibody1210/1211; (d) the heavy chain and/or the light chain of antibody1212/1213; (e) the heavy chain and/or the light chain of antibody2992/2993; (f) the heavy chain and/or the light chain of antibody2994/2995; (g) the heavy chain and/or the light chain of antibody2996/2993; (h) the heavy chain and/or the light chain of antibody2998/2999; (i) the heavy chain and/or the light chain of antibody3000/3001; (j) the heavy chain and/or the light chain of antibody3002/3003; (k) the heavy chain and/or the light chain of antibody3004/3005; (l) the heavy chain and/or the light chain of antibody3006/3007; or (m) the heavy chain and/or the light chain of antibody3008/3009.
 35. A polypeptide according to any of the preceding claimswherein binding domain B2 comprises the light chain variable region andthe heavy chain variable region of antibody 1208/1135 (SEQ ID NO: 7 andSEQ ID NO: 5), or a variant which has more than 60%, or more than 70%,e.g. 75 or 80%, preferably more than 85%, e.g. more than 90 or 95% aminoacid identity to SEQ ID NO: 7 and/or SEQ ID NO: 5).
 36. A polypeptideaccording to any one of claims 1 to 34 wherein binding domain B2comprises the light chain variable region and the heavy chain variableregion of antibody 1210/1211 (SEQ ID NO: 11 and SEQ ID NO: 9), or avariant which has more than 60%, or more than 70%, e.g. 75 or 80%,preferably more than 85%, e.g. more than 90 or 95% amino acid identityto SEQ ID NO: 11 and/or SEQ ID NO: 9).
 37. A polypeptide according toany one of claims 1 to 34 wherein binding domain B2 comprises the lightchain variable region and the heavy chain variable region of antibody2992/2993 (SEQ ID NO: 96 and SEQ ID NO: 97), or a variant which has morethan 60%, or more than 70%, e.g. 75 or 80%, preferably more than 85%,e.g. more than 90 or 95% amino acid identity to SEQ ID NO: 96 and/or SEQID NO: 97).
 38. A polypeptide according to any one of claims 1 to 34wherein binding domain B2 comprises the light chain variable region andthe heavy chain variable region of antibody 2994/2995 (SEQ ID NO: 98 andSEQ ID NO: 99), or a variant which has more than 60%, or more than 70%,e.g. 75 or 80%, preferably more than 85%, e.g. more than 90 or 95% aminoacid identity to SEQ ID NO: 98 and/or SEQ ID NO: 99).
 39. A polypeptideaccording to any one of the preceding claims wherein binding domain B1is an IgG and binding domain B2 is an scFv.
 40. A polypeptide accordingto any one of claims 1 to 38 wherein binding domain B1 is an scFv andbinding domain B2 is an IgG.
 41. A polypeptide according to any one ofclaims 1 to 38 wherein binding domain B1 is an scFv and binding domainB2 is an scFv (e.g. in an scFv₂-Fc format).
 42. A polypeptide accordingto any one of the preceding claims wherein: (a) B1 comprises the threeCDRs of the light chain and/or the three CDRs of the heavy chain ofantibody 1200/1201 (SEQ ID NOs: 54, 55 and 79 and/or SEQ ID NOs: 46, 65and 72) and B2 comprises the three CDRs of the light chain and/or thethree CDRs of the heavy chain of antibody 1208/1135 (SEQ ID NOs: 54, 55and 57 and/or SEQ ID NOs: 46, 48 and 51); (b) B1 comprises the threeCDRs of the light chain and/or the three CDRs of the heavy chain ofantibody 1200/1201 (SEQ ID NOs: 54, 55 and 79 and/or SEQ ID NOs: 46, 65and 72) and B2 comprises the three CDRs of the light chain and/or thethree CDRs of the heavy chain of antibody 1210/1211 (SEQ ID NOs: 54, 55and 58 and/or SEQ ID NOs: 46, 48 and 52); (c) B1 comprises the threeCDRs of the light chain and/or the three CDRs of the heavy chain ofantibody 1618/1619 (SEQ ID NOs: 54, 55 and 83 and/or SEQ ID NOs: 62, 69and 76) and B2 comprises the three CDRs of the light chain and/or thethree CDRs of the heavy chain of antibody 1208/1135 (SEQ ID NOs: 54, 55and 57 and/or SEQ ID NOs: 46, 48 and 51); or (d) B1 comprises the threeCDRs of the light chain and/or the three CDRs of the heavy chain ofantibody 1618/1619 (SEQ ID NOs: 54, 55 and 83 and/or SEQ ID NOs: 62, 69and 76) and B2 comprises the three CDRs of the light chain and/or thethree CDRs of the heavy chain of antibody 1210/1211 (SEQ ID NOs: 54, 55and 58 and/or SEQ ID NOs: 46, 48 and 52).
 43. A polypeptide according toany one of the preceding claims wherein: (a) B1 comprises the lightchain variable region and/or the heavy chain variable region of antibody1200/1201 (SEQ ID NO: 19 and/or SEQ ID NO: 17) and B2 comprises thelight chain variable region and/or the heavy chain variable region ofantibody 1208/1135 (SEQ ID NO: 7 and/or SEQ ID NO: 5); (b) B1 comprisesthe light chain variable region and/or the heavy chain variable regionof antibody 1200/1201 (SEQ ID NO: 19 and/or SEQ ID NO: 17) and B2comprises the light chain variable region and/or the heavy chainvariable region of antibody 1210/1211 (SEQ ID NO: 11 and/or SEQ ID NO:9); (c) B1 comprises the light chain variable region and/or the heavychain variable region of antibody 1618/1619 (SEQ ID NO: 35 and/or SEQ IDNO: 33) and B2 comprises the light chain variable region and/or theheavy chain variable region of antibody 1208/1135 (SEQ ID NO: 7 and/orSEQ ID NO: 5); or (d) B1 comprises the light chain variable regionand/or the heavy chain variable region of antibody 1618/1619 (SEQ ID NO:35 and/or SEQ ID NO: 33) and B2 comprises the light chain variableregion and/or the heavy chain variable region of antibody 1210/1211 (SEQID NO: 11 and/or SEQ ID NO: 9); or (e) B1 and/or B2 comprise variants ofsaid light chain variable regions and/or said heavy chain variableregions having at least 90% sequence identity thereto.
 44. A polypeptideaccording to any one of the preceding claims wherein B1 comprises thelight chain variable region and/or the heavy chain variable region ofantibody 1200/1201 (SEQ ID NO: 19 and/or SEQ ID NO: 17) and B2 comprisesthe light chain variable region and/or the heavy chain variable regionof antibody 1210/1211 (SEQ ID NO: 11 and/or SEQ ID NO: 9), or variantsof said light chain variable regions and/or said heavy chain variableregions (for example, having at least 90% sequence identity thereto), orvice versa.
 45. A polypeptide according to any one of the precedingclaims wherein B1 comprises the light chain variable region and/or theheavy chain variable region of antibody 1618/1619 (SEQ ID NO: 35 and/orSEQ ID NO: 33) and B2 comprises the light chain variable region and/orthe heavy chain variable region of antibody 1210/1211 (SEQ ID NO: 11and/or SEQ ID NO: 9), or variants of said light chain variable regionsand/or said heavy chain variable regions (for example, having at least90% sequence identity thereto), or vice versa.
 46. A polypeptideaccording to any one of the preceding claims comprising a heavy chainconstant region having an amino acid sequence of SEQ ID NO:94 or 96and/or a light chain constant region having an amino acid sequence ofSEQ ID NO:95.
 47. A polypeptide according to any one of the precedingclaims wherein the polypeptide comprises at least one of the following:(a) an Fc region comprising ‘LALA’ mutations; (b) an scFv comprisingmutations in the heavy and light chain variable regions to cysteineresidues capable of forming a disulphide bridge; and/or (c) an scFvcomprising mutation in the heavy chain variable region to create one ormore N-glycosylation sites.
 48. A polypeptide according to any one ofthe preceding claims comprising (a) a binding domain (B1) comprising aheavy chain variable region of any of SEQ ID NOs: 17, 21, 27, 29, 33,37, 41, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136 or138 and a light chain variable region of any of SEQ ID NOs: 19, 23, 25,31, 35, 39, 43, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135 or139; (b) a heavy chain constant region comprising an Fc region (forexample, SEQ ID NO: 94 or 96); (c) a binding domain (B2) comprising aheavy chain variable region of any of SEQ ID NOs: 1, 5, 9, 13, 96, 98,100, 102, 104, 106, 108, 110 or 112 and a light chain variable region ofany of SEQ ID NOs: 3, 7, 11, 15, 97, 99, 101, 103, 105, 107, 109, 111 or113; and (d) optionally, a light chain constant region (for example SEQID NO:95).
 49. An isolated nucleic acid molecule encoding a bispecificpolypeptide according to any one of the preceding claims, or a componentpolypeptide chain thereof.
 50. A nucleic acid molecule according toclaim 49 wherein the molecule is a cDNA molecule.
 51. A nucleic acidmolecule according to claim 49 or 50 encoding an antibody heavy chain orvariable region thereof.
 52. A nucleic acid molecule according to anyone of claims 49 to 51 encoding an antibody light chain or variableregion thereof.
 53. A vector comprising a nucleic acid moleculeaccording to any one of claims 49 to
 52. 54. A vector according to claim53 wherein the vector is an expression vector.
 55. A recombinant hostcell comprising a nucleic acid molecule according to any one of claims49 to 52 or a vector according to claim 53 or
 54. 56. A host cellaccording to claim 55 wherein the host cell is a bacterial cell.
 57. Ahost cell according to claim 55 wherein the host cell is a mammaliancell.
 58. A host cell according to claim 55 wherein the host cell is ahuman cell.
 59. A method for producing bispecific polypeptide accordingto any one of the claims 1 to 48, the method comprising culturing a hostcell as defined in any of claims 55 to 58 under conditions which permitexpression of the bispecific polypeptide or component polypeptide chainthereof.
 60. A pharmaceutical composition comprising an effective amountof bispecific polypeptide according to any one of the claims 1 to 48 anda pharmaceutically-acceptable diluent, carrier or excipient.
 61. Apharmaceutical composition according to claim 60 adapted for parenteraldelivery.
 62. A pharmaceutical composition according to claim 60 adaptedfor intravenous delivery.
 63. A bispecific polypeptide according to anyone of the claims 1 to 48 for use in medicine.
 64. A bispecificpolypeptide according to any one of the claims 1 to 48 for use intreating or preventing a neoplastic disorder in a subject.
 65. Apolypeptide for use according to claim 64 wherein the neoplasticdisorder is associated with the formation of solid tumours within thesubject's body.
 66. A polypeptide for use according to claim 65 whereinthe solid tumour is selected from the group consisting of prostatecancer, breast cancer, lung cancer, colorectal cancer, melanomas,bladder cancer, brain/CNS cancer, cervical cancer, oesophageal cancer,gastric cancer, head/neck cancer, kidney cancer, liver cancer,lymphomas, ovarian cancer, pancreatic cancer and sarcomas.
 67. Apolypeptide for use according to claim 66 wherein the solid tumour isselected from the groups consisting of renal cell carcinoma, colorectalcancer, lung cancer, prostate cancer and breast cancer.
 68. Apolypeptide for use according to any one of claims 64 to 67 wherein thepolypeptide is for use in combination with one or more additionaltherapeutic agents.
 69. A polypeptide for use according to claim 68wherein the one or more additional therapeutic agents is/are animmunotherapeutic agent that binds a target selected from the groupconsisting of PD-1/PD-1L, CTLA-4, OX40, CD40, GITR, LAG3, TIM3, CD27 andKIR.
 70. Use of a bispecific polypeptide according to any one of theclaims 1 to 46 in the preparation of a medicament for treating orpreventing a neoplastic disorder in a subject.
 71. A use according toclaim 70 wherein the neoplastic disorder is associated with theformation of solid tumours within the subject's body.
 72. A useaccording to claim 71 wherein the solid tumour is selected from thegroup consisting of prostate cancer, breast cancer, lung cancer,colorectal cancer, melanomas, bladder cancer, brain/CNS cancer, cervicalcancer, oesophageal cancer, gastric cancer, head/neck cancer, kidneycancer, liver cancer, lymphomas, ovarian cancer, pancreatic cancer andsarcomas.
 73. A use according to claim 72 wherein the solid tumour isselected from the groups consisting of renal cell carcinoma, colorectalcancer, lung cancer, prostate cancer and breast cancer.
 74. A useaccording to any one of claims 70 to 73 wherein the polypeptide is foruse in combination with one or more additional therapeutic agents.
 75. Apolypeptide for use according to claim 74 wherein the one or moreadditional therapeutic agents is/are an immunotherapeutic agent thatbinds a target selected from the group consisting of PD-1/PD-1L, CTLA-4,OX40, CD40, GITR, LAG3, TIM3, CD27 and KIR.
 76. A method for thetreatment or diagnosis of a neoplastic disorder in a subject, comprisingthe step of administering to the subject an effective amount of abispecific polypeptide according to any one of the claims 1 to
 48. 77. Amethod according to claim 76 wherein the neoplastic disorder isassociated with the formation of solid tumours within the subject'sbody.
 78. A method according to claim 77 wherein the solid tumour isselected from the group consisting of prostate cancer, breast cancer,lung cancer, colorectal cancer, melanomas, bladder cancer, brain/CNScancer, cervical cancer, oesophageal cancer, gastric cancer, head/neckcancer, kidney cancer, liver cancer, lymphomas, ovarian cancer,pancreatic cancer and sarcomas.
 79. A method according to claim 78wherein the solid tumour is selected from the groups consisting of renalcell carcinoma, colorectal cancer, lung cancer, prostate cancer andbreast cancer.
 80. A method according to any one of claims 76 to 79wherein the subject is human.
 81. A method according to any one ofclaims 76 to 80 wherein the method comprises administering thebispecific antibody systemically.
 82. A method according to any one ofclaims 76 to 81 further comprising administering to the subject one ormore additional therapeutic agents.
 83. A method according to any one ofclaims 76 to 82 wherein the one or more additional therapeutic agentsis/are an immunotherapeutic agent that binds a target selected from thegroup consisting of PD-1/PD-1L, CTLA-4, OX40, CD40, GITR, LAG3, TIM3,CD27 and KIR.
 84. A bispecific polypeptide substantially as describedherein with reference to the description and figures.
 85. Apolynucleotide substantially as described herein with reference to thedescription and figures.
 86. A pharmaceutical composition substantiallyas described herein with reference to the description and figures. 87.Use of a bispecific polypeptide substantially as described herein withreference to the description and figures.
 88. A method of treatmentsubstantially as described herein with reference to the description andfigures.